Dietary flavonoids interact with trace metals and affect metallothionein level in human intestinal cells

Flavonoids are natural compounds found in food items of plant origin. The study examined systematically the interaction of structurally diverse dietary flavonoids with trace metal ions and the potential impact of dietary flavonoids on the function of intestinal cells. Spectrum analysis was first performed to determine flavonoid-metal interaction in the buffer. Among the flavonoids tested, genistein, biochanin-A, naringin, and naringenin did not interact with any metal ions tested. Members of the flavonol family, quercetin, rutin, kaempferol, flavanol, and catechin, were found to interact with Cu(II) and Fe(III). On prolonged exposure, quercetin also interacted with Mn(II). Quercetin at 1:1 ratio to Cu(II) completely blocked the Cu-dependent color formation from hematoxylin. When quercetin was added to the growth medium of cultured human intestinal cells, Caco-2, the level of metal binding antioxidant protein, metallothionein, decreased. The effect of quercetin on metallothionein was dose- and time-dependent. Genistein and biochanin A, on the contrary, increased the level of metallothionein. The interaction between dietary flavonoids and trace minerals and the effect of flavonoids on metallothionein level imply that flavonoids may affect metal homeostasis and cellular oxidative status in a structure-specific fashion.     

Pathways of glutathione metabolism and transport in isolated proximal tubular cells from rat kidney

Cellular uptake and metabolism of exogenous glutathione (GSH) in freshly isolated proximal tubular (PT) cells from rat kidney were examined in the absence and presence of inhibitors of GSH turnover [acivicin, L-buthionine-S,R-sulfoximine (BSO)] to quantify and assess the role of different pathways in the handling of GSH in this renal cell population. Incubation of PT cells with 2 or 5 mM GSH in the presence of acivicin/BSO produced 3- to 4-fold increases in intracellular GSH within 10-15 min. These significantly higher intracellular concentrations were maintained for up to 60 min. At lower concentrations of extracellular GSH, an initial increase in intracellular GSH concentrations was observed, but this was not maintained for the 60-min time course. In the absence of inhibitors, intracellular concentrations of GSH increased to levels that were 2- to 3-fold higher than initial values in the first 10-15 min, but these dropped below initial levels thereafter. In both the absence and presence of acivicin/BSO, PT cells catalyzed oxidation of GSH to glutathione disulfide (GSSG) and degradation of GSH to glutamate and cyst(e)ine. Exogenous tert-butyl hydroperoxide oxidized intracellular GSH to GSSG in a concentration-dependent manner and extracellular GSSG was transported into PT cells, but limited intracellular reduction of GSSG to GSH occurred. Furthermore, incubation of cells with precursor amino acids produced little intracellular synthesis of GSH, suggesting that PT cells have limited biosynthetic capacity for GSH under these conditions. Hence, direct uptake of GSH, rather than reduction of GSSG or resynthesis from precursors, may be the primary mechanism to maintain intracellular thiol redox status under toxicological conditions. Since PT cells are a primary target for toxicants, the ability of these cells to rapidly take up and metabolize GSH may serve as a defensive mechanism to protect against chemical injury.     

Alterations in corticotropin-releasing factor and vasopressin content in rat brain during morphine withdrawal: correlation with hypothalamic noradrenergic activity and pituitary-adrenal response

Oxidative stress is thought to play an important role in the pathogenesis of Parkinson's disease (PD). Glutathione (GSH), a major cellular antioxidant, is decreased in the substantia nigra pars compacta of PD patients. The aim of the present study was to investigate whether deprenyl and its desmethyl metabolite, putative neuroprotective agents in the treatment of PD, could protect cultured rat mesencephalic neurons from cell death caused by GSH depletion due to treatment with L-buthionine-(S,R)-sulfoximine (BSO). BSO (10 microM) caused extensive cell death after 48 hr, as demonstrated by disruption of cellular integrity and release of lactate dehydrogenase into the culture medium. Both deprenyl and desmethylselegiline, at concentrations of 5 and 50 microM, significantly protected dopaminergic neurons from toxicity without preventing the BSO-induced loss in GSH. Protection was not associated with monoamine oxidase type B inhibition in that pargyline, a potent MAO inhibitor, was ineffective and pretreatment with pargyline did not prevent the protective effects of deprenyl. Protection was not associated with inhibition of dopamine uptake by deprenyl because the dopamine uptake inhibitor mazindol did not diminish BSO toxicity. The antioxidant ascorbic acid (200 microM) also protected against BSO-induced cell death, suggesting that oxidative events were involved. This study demonstrates that deprenyl and its desmethyl metabolite can diminish cell death associated with GSH depletion.     

The MGDS examination: a systematic approach. 3. Part II of the examination: diagnosis, treatment planning, execution of treatment, maintenance and appraisal, writing-up log diaries

The purpose of this study was to elucidate the differential contribution of catalase and glutathione peroxidase (GSH-Px) to H2O2 scavenging in cultured human dermal fibroblasts. Responses of the cells in terms of both enzyme activities were examined by using two sorts of inhibitors, 3-amino-1H-1,2,4-triazole (AT) for catalase and DL-buthionine-[S,R]-sulfoximine (BSO) for GSH-Px, under exposure to H2O2 or ultraviolet (UV) B radiation. AT treatment resulted in a decrease in H2O2 scavenging activity, while BSO treatment did not affect H2O2 scavenging. When fibroblasts were exposed to a low concentration of H2O2 (100 microM). AT treatment resulted in a significant decrease in cell survival, but BSO treatment did not affect survival. At higher concentrations of H2O2 ranging from 500 microM to 1 mM, BSO-treated fibroblasts showed reduced survival. In addition, AT treatment was much more cytotoxic in the presence of UVB than BSO treatment. The intracellular levels of H2O2 in fibroblasts treated with AT or BSO were also determined. BSO-treated cells showed similar H2O2 levels to control cells, but the intracellular H2O2 levels of AT-treated fibroblasts were 1.4-fold higher than found in control cells. These results with human dermal fibroblasts indicate that catalase acts as a primary defence against oxidative stress from exogenous or endogenous H2O2 at low concentrations. In contrast, GSH-Px helps protect the cell from damage during exposure to high concentrations of H2O2.     

Comparative protection against oxyradicals by three flavonoids on cultured endothelial cells

Oxygen-derived free radicals are known to injure the endothelium of aorta in diverse disorders. In this study we compared the cytoprotective effects of three flavonoids against oxyradical damage to porcine aortic endothelial cells in vitro. Cultured porcine aortic endothelial cells were exposed to oxyradicals generated by xanthine oxidase--hypoxanthine (XO-HP). The cytoprotective activities of morin, quercetin, and catechin on these systems were compared using established morphologic criteria. The results in the XO-HP system showed that morin at 0.125, 0.25, and 0.5 mM delayed cell necrosis to 27.4 +/- 1.3, 46.8 +/- 1.8, and longer than 70 min, respectively, compared with 12.0 +/- 1.3 min in the control group. These degrees of protection were significantly stronger than those provided by quercetin and catechin at corresponding concentrations (p < 0.01). Morin and quercetin were moderate inhibitors of xanthine oxidase on the basis of the oxygen consumption rate, whereas catechin at the same concentrations had little inhibitory effect. The data from uric acid formation and cytochrome c reduction were consistent with the oxygen consumption measurement for the three flavonoids.     

Antiproliferative potency of structurally distinct dietary flavonoids on human colon cancer cells

Dietary flavonoids are known to be antiproliferative and may play an important role in cancer chemoprevention, especially cancers of the gastrointestinal tract, because of a direct contact with food. This study was designed to compare the antiproliferative potency of several structurally distinct dietary flavonoids in colon cancer cells, Caco-2 and HT-29, and in rat non-transformed intestinal crypt cells, IEC-6. Flavonoids varied significantly in their antiproliferative potency depending on the structural features but the observations were consistent among the three cell lines studied. Of the two most potent flavonoids, quercetin and genistein, the effect was found to be dose-dependent and chromatin condensation, an indication of apoptosis, was noticed. Quercetin was found to distribute throughout the cell with higher amounts in the perinuclear and nucleoli areas. The lack of specific cell membrane enrichment by quercetin was consistent with its lack of effect on the transepithelial resistance. While several flavonoids including quercetin were found to be unstable, the chemical instability did not correlate with the antiproliferative potency, although it may contribute to the antiproliferative effect.     

Quercetin, a bioflavonoid, inhibits the DNA synthesis of human leukemia cells

Quercetin, a flavonoid, is found in many plants, including edible fruits and vegetables. It has been proposed that flavonoids may have potential as anticancer agents. To test an aspect of this hypothesis, we examined the effects of the flavonoid, quercetin, on the DNA synthesis of the human leukemia cell, HL-60. Quercetin induced a dose-dependent inhibition of DNA synthesis in the test range of 1 microM to 1 mM. The inhibitory effect on DNA synthesis was evident as early as 24 h after the addition of quercetin. At the concentrations of 10 microM, 100 microM and 1 mM, 50, 82 and 85% of DNA synthesis, respectively, was inhibited by quercetin as compared to the control. After 48 and 72 h incubation of the cells with 100 microM and 1 mM quercetin, DNA synthesis was almost completely abolished. These results suggest that the inhibitory effects of quercetin on HL-60 cell DNA synthesis is not due to a non-specific cytotoxic effect, since following removal of quercetin, the treated cells regrew normally.     

Esophagitis in Sprague-Dawley rats is mediated by free radicals

Free radical-mediated esophagitis was studied during duodenogastroesophageal reflux (mixed reflux) or acid reflux in rats. The influence of reflux on esophageal glutathione levels was also examined. Mixed reflux caused more gross mucosal injury than acid reflux. Gross mucosal injury occurred in the mid-esophagus. Total glutathione (GSH) in the esophageal mucosa of control rats was highest in the distal esophagus. The time course of esophageal GSH in rats treated by mixed reflux showed a significant decrease 4 hr after initiation of reflux, followed by a significant increase from the 12th hour on. Mucosal GSH was increased in both reflux groups after 24 hr but significantly more so in the mixed than in the acid reflux group. The free radical scavenger superoxide dismutase (SOD) prevented esophagitis and was associated with decreased GSH levels. GSH depletion by buthionine sulfoximine (BSO) prevented esophagitis and stimulated SOD production in the esophageal mucosa. It is concluded that gastroesophageal reflux is associated with oxidative stress in the esophageal mucosa. The lower GSH levels in the mid-esophagus may predispose to damage in this area. Duodenogastroesophageal reflux causes more damage than pure acid reflux. Oxidative stress leads to GSH depletion of the esophageal mucosa in the first few hours following damage but then stimulates GSH production. GSH depletion by BSO does not worsen esophagitis since it increases the esophageal SOD concentration.     

Caveolae: from a morphological point of view

The protective effect of quercetin against oxidant-induced cell injury (hypoxanthine/xanthine oxidase system) was studied in the renal tubular epithelial cell line LLC-PK1. Pretreatment with quercetin provided protection from structural and functional cell damage in a concentration-dependent manner (10-100 microM). Comparison with structural variants revealed that the protective property of quercetin depends on the number of hydroxyl substituents in the B-ring, the presence of an extended C-ring chromophore, 3-D-planarity and lipophilicity, indicating that membrane affinity is essential for protection. The hypothesis that quercetin exerts its protective effects via inhibition of lipid peroxidation was further examined. Protection by quercetin was found when lipid peroxidation, assessed by the release of malondialdehyde, was initiated by H2O2 or by the combination of 1-chloro-2,4-dinitrobenzene and aminotriazole. In contrast, the bioflavonoid was not protective when oxidative cell damage was induced by menadione and occurred in the absence of lipid peroxidation. These data suggest that cytoprotective effects of quercetin are related to membrane affinity and may be explained by interruption of membrane lipid peroxidation rather than by intracellular scavenging of oxygen free radicals.     

Establishment of a novel human myeloid leukaemia cell line (FKH-1) with t(6;9)(p23;q34) and the expression of dek-can chimaeric transcript

Calcein-labeled B16 melanoma (B16M) cells were injected intraportally, and in vivo video microscopy was used to study the distribution and damage of cancer cells arrested in the liver microvasculature over a period of 4 hours. The contribution of glutathione (GSH)-dependent antioxidant machinery to the possible oxidative stress-resistance mechanism of B16M cell was determined by in vitro incubation with the selective inhibitor of GSH synthesis L-buthionine (S,R)-sulphoximine (BSO) before B16M cell injection in untreated and 0.5-mg/kg lipopolysaccharide (LPS)-treated mice. In addition, untreated and LPS-treated isolated syngeneic hepatic sinusoidal endothelial cells (HSE) were used to determine in vitro their specific contribution to B16M cell damage. Trauma inherent to intrasinusoidal lodgement damaged 35% of B16M cells in both normal and LPS-treated mouse liver. The rest of the arrested B16M cells remained intact in normal liver for at least 4 hours, although their damaged cell percentage significantly (P < .05) increased since the second hour in normal mice injected with BSO-treated cells and since the first hour in LPS-treated mice given untreated cells. Recombinant human interleukin-1 receptor antagonist (rHuIL-1-Ra) given to mice 15 minutes before LPS significantly (P < .05) abrogated B16M cell damage. On the other hand, 40% of the B16M cells co-cultured with unstimulated HSE and 70% of the co-cultured with LPS-treated HSE became sensitive to endothelial cell-mediated damage after BSO treatment. These results demonstrate that a high intracellular level of GSH protects B16M cells from possible in vivo and in vitro sinusoidal cell-mediated oxidative stress, contributing to the mechanism of metastatic cell survival within the hepatic microvasculature.     

Modulation of intracellular glutathione and cysteine metabolism in bovine oviduct epithelial cells cultured In vitro

The objective of this study was to determine how alterations in intracellular thiol levels of oviduct epithelium occur in response to chemical or environmental conditions that could result in oxidative stress. Bovine oviducts were classified as follicular (F) or luteal (L) according to the reproductive stage of the ovary. Epithelial cells were harvested from the ampulla (AMP) and isthmus (ISTH) region of each oviduct, suspended in culture medium, and then plated into collagen-coated culture plates and grown to confluency. Basal levels of intracellular cysteine (Cys) and glutathione (GSH) were determined in oviduct epithelial cells and found to range from 0.36 to 0.46 pmol/ microg protein for Cys and from 5.3 to 6.4 pmol/ microg protein for GSH. Oxidized Cys values ranged from 21% to 39% of total Cys, whereas the oxidized GSH levels observed were from 21% to 28% of total GSH except in luteal ISTH, where they were significantly lower (6%). Confluent cells were exposed to GSH-depleting agents, L-buthionine-S,R-sulfoximine (BSO) or diethyl maleate (DEM), at doses ranging from 10 to 5000 microM. Both compounds depleted GSH in a dose-dependent manner, and 500 microM concentrations were chosen for subsequent studies with each compound. Cys levels in BSO (500 microM)-treated oviduct epithelial cells were transiently elevated over control values during the initial 5-h incubation; there was then a decrease in Cys levels by AMP but not ISTH oviduct epithelial cells. BSO-treated oviduct epithelial cells displayed a continued depletion of GSH over the incubation period and by 24 h were depleted by 38% to 61%. These results demonstrate a difference in GSH turnover in oviduct epithelial cells associated with reproductive stage. Exposure to DEM (500 microM) caused a decline in both Cys and GSH levels, which were partially restored after DEM removal. In general, L-staged oviduct epithelial cells were observed to be more competent at replenishing thiol stores than F-staged oviduct epithelial cells. Results from this study suggest that reproductive stage and region influence intracellular oviduct epithelium thiol status and therefore may affect how this tissue responds to chemicals or environmental conditions leading to oxidative stress.     

Spatial learning and memory deficits induced by dopamine administration with decreased glutathione

Administration of buthionine sulfoximine (BSO) selectively inhibits glutathione (GSH) biosynthesis and induces a GSH deficiency. Decreased GSH levels in the brain may result in less oxidative stress (OS) protection, because GSH contributes substantially to intracellular antioxidant defense. Under these conditions, administration of the pro-oxidant, dopamine (DA), which rapidly oxidizes to form reactive oxygen species, may increase OS. To test the cognitive behavioral consequences of decreased GSH, BSO (3.2 mg in 30 microliters, intracerebroventricularly) was administered to male Fischer 344 rats every other day for 4 days. In addition, DA (15 microliters of 500 microM) was administered every day [either 1 h after BSO (BSO + DA group) or 1 h before BSO (DA + BSO group), when given on the same day as BSO] and spatial learning and memory assessed (Morris water maze, six trials/day). BSO + DA rats, but not DA + BSO rats, demonstrated cognitive impairment compared to a vehicle group, as evidenced by increased latencies to find the hidden platform, particularly on the first trial each day. Also, the BSO + DA group utilized non-spatial strategies during the probe trials (swim with no platform): i.e., less time spent in the platform quadrant, fewer crossings and longer latencies to the previous platform location, and more time spent in the platform quadrant, fewer crossings and longer latencies to the previous platform location, and more time spent around the edge of the pool rather than in the platform zone. Therefore, the cognitive behavioral consequences of decreasing GSH brain levels with BSO in conjunction with DA administration depends on the order of administration. These findings are similar to those seen previously on rod and plank walking performance, as well as to those seen in aged rats, suggesting that the oxidation of DA coupled with a reduced capacity to respond to oxidative stress may be responsible for the induction of age-related cognitive deficits.     

Women and menopause: beliefs, attitudes, and behaviors. The North American Menopause Society 1997 Menopause Survey

It has been demonstrated that exposure to mercury or cadmium compounds causes alterations in the glutathione system in a model glial cell line, C6. Here we report that two organic tin compounds, triethyltin (TET) and trimethyltin (TMT), are also toxic to these cells with EC50 values for cell death of c. 0.02 microM and 0.8 microM respectively. Exposure for 24 h to either of these compounds at sub-toxic concentrations caused increases in the amount of reduced glutathione (GSH) per cell. Increases in glutathione-S-transferase enzyme activity were also demonstrated after TET or TMT exposure. This suggests that glutathione increases occur in glial cells after toxic insults below that required to cause cell death, possibly acting as a protective mechanism. To test whether GSH plays a role in organotin-induced cell death we manipulated GSH in the culture media or via intracellular GSH and looked at the effects on sensitivity to TET or TMT toxicity. Adding GSH to the culture media did not protect the cells. Depletion of intracellular GSH with buthionine-[S,R] sulphoximine did not alter cytotoxicity of TET or TMT. However, pre-treatment with (-)-2-oxo-4-thiazolidine carboxylic acid (OTC), which increases intracellular GSH levels, protected the cells against both compounds. The EC50 for TMT was increased from 0.77 to 1.8 microM, a 2.3-fold shift, whereas the EC50 for TET was increased > 20-fold, from 0.022 to 0.47 microM. One interpretation of these results is that GSH protects cells against the toxicity of organic tin compounds without reacting directly with them to any significant extent. Under conditions where GSH is depleted, additional protective mechanisms may be active.     

Psychomotor effects of dopamine infusion under decreased glutathione conditions

Administration of buthionine sulfoximine (BSO) selectively inhibits glutathione (GSH) biosynthesis, thereby inducing a GSH deficiency. Because GSH plays a critical role in intracellular antioxidant defense, decreased GSH levels in the brain may result in less oxidative stress (OS) protection. Thus, the pro-oxidant effects of dopamine (DA), which rapidly oxidizes to form reactive oxygen species, may increase. In this study, the behavioral consequences of reduced OS protection were examined by administering BSO (3.2 mg in 30 microl Ringer's solution, intracerebroventricularly) every other day for 12 d to male Fischer 344 rats. In addition, DA (15 microl of 500 microM) was administered every day; when given on the same day as BSO, it was either 1 h after BSO (BSO + DA group) or 1 h before BSO (DA + BSO group). Tests of psychomotor behavior--rod walking, wire suspension, and plank walking--were performed five times during the experiment. BSO + DA administration, but not DA + BSO, impaired performance by decreasing latency to fall in the rod and plank walk tests compared to a vehicle only (Ringer's) group. Therefore, depletion of GSH with BSO, followed by DA treatment, produced deficits in psychomotor behavior. These deficits are similar to those seen in aged rats, suggesting that the oxidation of DA coupled with a reduced capacity to respond to OS may be responsible for the induction of age-related motor behavioral deficits.     

Point-of-care (POC) measurement of coagulation after cardiac surgery

Infection of feline immunodeficiency virus (FIV) has been shown to induce apoptosis that might be associated with the lymphocyte depletion in the infected cats. To investigate the inhibitory effect of antioxidants on FIV-induced apoptosis, we examined the effect of N-acetylcysteine (NAC) and ascorbic acid (AA) on apoptosis and virus replication in feline lymphoblastoid (Fel-039) and fibroblastoid (CRFK) cell lines infected with FIV. The treatment with NAC or AA induced a significant inhibition of viral replication and apoptosis in Fel-039 cells and tumor necrosis factor alpha (TNF-alpha)-treated CRFK cells infected with FIV. Both cell lines in the presence of noncytotoxic concentrations of NAC or AA showed in increase of intracellular glutathione (GSH) level, which might protect the cells against oxidative stresses exerted by FIV infection and TNF-alpha treatment. On the basis of these in vitro results, we suggest that antioxidant therapies aimed at restoring depleted GSH level might be effective for inhibition of viral replication and cell death associated with the development of immunodeficiency.     

Synergistic action of quercetin and genistein in human ovarian carcinoma cells

Ovarian carcinoma is the fourth most common cause of cancer death in women and there has been a steady increase in the age-adjusted cancer death rates in the past 25 years in the US. However, patients who become cisplatin resistant respond poorly to available cytotoxic agents; therefore, discovering novel targets for ovarian carcinoma is vital. Quercetin, an anticancer agent, arrests the cell cycle at G1 and S phase boundary. Genistein, a plant flavonoid, attacks the cell cycle at G2 and/or early M phases in most carcinoma cells. Quercetin and genistein block the phosphatidylinositol conversion to IP3 signal transduction pathway mainly by inhibiting 1-phosphatidylinositol 4-kinase (PI kinase, EC 2.7.1.67) and 1-phosphatidylinositol 4-phosphate 5-kinase (PIP kinase, EC 2.7.1.68), respectively. Because each drug attacks a different phase of the cell cycle and reduces IP3 concentration by attacking different signal transduction enzymes, we tested the hypothesis that the two drugs might be synergistic in human carcinoma cells. In human ovarian carcinoma OVCAR-5 cells in growth inhibition assay, the IC50S for quercetin and genistein were (mean +/- SE) 66 +/- 3.0 and 32 +/- 2.5 microM; in clonogenic assays they were 15 +/- 1.2 and 5 +/- 0.5 microM, respectively. When quercetin was added to the cultures of OVCAR-5 cells followed 8 h later by genistein, synergism was observed in growth inhibition and clonogenic assays. The synergistic action of quercetin and genistein may be of interest in clinical treatment of human ovarian carcinoma.     

Direct evidence for glutathione as mediator of apoptosis in neuronal cells

Recent evidence has focused attention on the role of oxidative stress in various acute and chronic neurodegenerative diseases. Particularly, a decrease in the level of the powerful antioxidant glutathione (GSH) and death of dopaminergic neurons in substantia nigra are prominent features in Parkinson's disease. The mode of neuronal death is uncertain; however, apoptosis has been hypothesized to be mediated through the induction of free radicals via oxidative pathways. An approach to determine the role of GSH depletion in neurodegeneration and apoptosis was to create a selective modulation of this antioxidant by metabolic manipulations in a clonal cell line of neuronal origin (mouse neuroblastoma NS20Y). Intracellular GSH levels was lowered by inhibiting its biosynthesis with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. This treatment led to a GSH depletion of 50% after 1 h and 98% after 24 h. A direct cause/effect relationship between GSH depletion and apoptosis was evidenced in this neuronal cell type. GSH depletion induced the death of NS20Y and promoted nuclear alterations of apoptosis as demonstrated by the in situ staining of DNA fragmentation after 5 days of BSO treatment (by terminal-deoxynucleotide transferase-mediated dUTP-nick end labeling), and the appearance of DNA laddering on agarose gel. These results suggested that redox desequilibrium induced by GSH depletion may serve as a general trigger for apoptosis in neuronal cells, and are consistent with the hypothesis that GSH depletion contribute to neuronal death in Parkinson's disease.     

Combined effects of buthionine sulfoximine and cepharanthine on cytotoxic activity of doxorubicin to multidrug-resistant cells

We studied the potentiation of doxorubicin (DOX) activity in multidrug-resistant (MDR) cells by buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, and by cepharanthine (CE), which interacts with P-glycoprotein. The glutathione (GSH) of MDR cells was approximately 1.5-fold greater than that of the parental cell line. BSO reduced GSH content of MDR cells compared to that of the sensitive ones. The BSO treatment (50 microM) enhanced the effect of DOX by 1.8-fold, while CE caused a greater reversal of drug resistance. The combination of BSO with CE produced further potentiation of DOX activity in an antiproliferative effect. Pretreatment of cells with BSO did not alter the cellular accumulation of DOX in the absence or presence of CE. The addition of BSO (30 mM) to the drinking water of mice reduced the tissue levels of GSH in tumor cells, suggesting that the marked decrease in GSH might diminish the ability of that tumor to resist DOX. Combined administration of CE and DOX resulted in enhancement of DOX antitumor activity and prolongation of survival time. The survival of mice treated with BSO and CE as a supplement to DOX treatment was superior that of mice receiving DOX alone. These studies demonstrated that the combinations of BSO with CE may be useful for killing drug-resistant tumor cells.     

Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product

In the last ten years, there has been an important increase in interest in quercetin action as a unique antioxidant, but its putative role in numerous prooxidant effects is also being continually updated. The mechanism underlying this undesirable ability seems to involve its metabolic oxidoreductive activation. Based on the structural properties of quercetin, we have investigated whether its catechol moiety may be the potential tool for revealed toxicity. We demonstrated, with an ESR spin-stabilization technique coupled to conventional spectrophotometry, that o-semiquinone and o-quinone are indeed the products of enzymatically catalyzed oxidative degradation of quercetin. The former radical might serve to facilitate the formation of superoxide and depletion of GSH, which could confer a specificity of its prooxidative action in situ. The observed one-electron reduction of o-quinone may enrich the semiquinone pool, thereby magnifying its effect. The two-electron reduction of quinone can result in constant resupply of quercetin in situ, thereby also modulating another pathway of its known biological activities. We have also tried to see whether the intracellular oxidative degradation of quercetin can be confirmed under the controlled conditions of model monolayer cell cultures. The results are indicative of the intracellular metabolic activation of quercetin to o-quinone, the process which can be partially associated with the observed concentration-dependent cytotoxic effect of quercetin.