Metabolic responses induced by thrombin in human umbilical vein endothelial cells

Metabolic responses induced by thrombin in human umbilical vein endothelial cells (HUVECs) were investigated by using the cytosensor technique. Thrombin increased the extracellular acidification rate of endothelial cells, measured as an index of metabolic activity with a cytosensor microphysiometer, in a concentration-dependent fashion with an EC(50) of 1.27+/-0.59 IU/ml, which was abolished by the MAP kinase inhibitor PD98059. When intracellular Ca(2+) was chelated or PKC was inactivated, PD98059 failed to abolish the thrombin-induced acidification rate response in HUVECs. In addition, the tyrosine kinase inhibitor genistein, PKC inhibitor calphostin C, and Na(+)/H(+)exchanger antagonist MIA also partly inhibited thrombin-induced acidification rate responses. It is suggested that thrombin stimulated rapid metabolic responses via MAP kinase in HUVECs, which are calcium- and PKC-dependent.     

In Vitro Studies of the Metabolism of Tissue Slices : I. Metabolic and Physical Factors Influencing the Penetration of Tetrazolium Salts

The use of tetrazolium salts for metabolic studies has been dismissed on the basis of their poor penetration into fresh tissue slices. In view of the fact that the penetration of these compounds can be visualized, it was felt that knowledge of the factors involved would be important. Factors, known to influence the penetration of oxygen, were examined with respect to the tetrazolium salts. The penetration of tetrazolium salt into tissue slices was a regular and predictable phenomenon. It was found that decreasing the metabolism of the cells in the slice substantially increased the penetration of these compounds, while increases in metabolism, by addition of substrate (such as succinate) to the incubating medium, considerably decreased their penetration. Increasing concentrations of the salt in the medium resulted in greater but limited penetration. It is our belief that the metabolism of tissue slices can be effectively studied with the aid of the tetrazolium salts, the portion of the population of cells participating in any reaction being accurately established by measuring the depth of the zone of reduced dye.     

Effect of esculetin on activities of pumpkin glutathione S-transferases and growth of pumpkin seedlings

The effect of esculetin and some related coumarins (coumarin, 7-hydroxycoumarin and scopoletin) on growth of pumpkin (Cucurbita maxima Duch.) seedlings and the activities of pumpkin glutathione S-transferases (GSTs) were investigated. Coumarin and esculetin affected the growth of seedlings. The hypocotyls of affected seedlings became weak and bent at the mid region, roots became very soft with brownish discoloration, and finally seedlings died. Among the compounds tested, only esculetin inhibited CmGSTU3 and CmGSTU2 activities measured with 1-chloro-2,4-dinitrobenzene (CDNB) and at a concentration of 22 μM, it inhibited the activity of CmGSTU3 by 50 %.     

Inhibition of the formation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid from arachidonic acid in polymorphonuclear leukocytes by various coumarins

The effects of various coumarins (i.e. esculetin, daphnetin and fraxetin) on the formation of the 5-lipoxygenase product, 5-HETE, and the cyclooxygenase product, HHT, were studied. Esculetin (6,7-dihydroxycoumarin) was found to inhibit the formation of 5-HETE more strongly than HHT; its concentrations for 50% inhibition (IC50) were 1.46 +/- 1.02 microM for the formation 5-HETE and 57.3 +/- 17.3 microM for the formation of HHT. Daphnetin (7,8-dihydroxycoumarin) and fraxetin (6-methoxy-7,8-dihydroxycoumarin) also inhibited the formation of the 5-lipoxygenase product, 5-HETE, and the cyclooxygenase product, HHT; their IC50 values were, respectively, 6.90 +/- 2.07 microM and 2.57 +/- 0.088 microM for the formation of 5-HETE and 139.0 +/- 30.0 microM and 532.5 +/- 33.0 microM for the formation of HHT. The monohydroxy coumarin derivatives umbelliferone (7-hydroxycoumarin) and scopoletin (6-methoxy-7-hydroxycoumarin) and the coumarin glucosides fraxin (6-methoxy-7,8-dihydroxycoumarin 8-O-D-glucoside) and esculin (6,7-dihydroxycoumarin 6-O-D-glucoside) also inhibited the formation of 5-HETE, though less strongly. 4-Hydroxycoumarin and coumarin had no effect on either 5-lipoxygenase or cyclooxygenase at concentrations of up to 1 mM. Esculetin inhibited the formation of 5-HETE noncompetitively. In contrast, the dimethoxycoumarin fraxidin (6,8-dimethoxy-7-hydroxycoumarin) inhibited the formation of HHT more strongly than the formation of 5-HETE at a concentration of 1 mM.     

Screening for novel drug effects with a microphysiometer: A potent effect of clofilium unrelated to potassium channel blockade

Changes in cellular metabolism in response to pharmacological compounds can be detected using a biosensor known as a microphysiometer, which measures the rate at which cells release acidic metabolites. We have applied this technique to screen for effects of cation channel blockers on the metabolism of a variety of human and murine cell lines. At concentrations sufficient for cation channel blockade, most of these drugs have little or no effect on cellular metabolism as measured by acid release. In contrast, the potassium channel blocker clofilium triggers sustained increases in acid release at low (≥3 μM) concentration. Acid release persists in media containing high (150 mM) extracellular potassium. This release is not triggered by chemically similar potassium channel blockers. Thus these metabolic effects reflect a potent and specific function of clofilium which is unrelated to potassium channel blockade. Attempts to identify physiological correlates to this response revealed that low concentrations of clofilium but not other potassium channel blockers cause lymphoma apoptosis. These findings demonstrate that effects of clofilium found in other studies may not be due to changes in plasma membrane potassium conductance.     

gAd-globular head domain of adiponectin increases fatty acid oxidation in newborn rabbit hearts

Adiponectin is an adipocyte-derived hormone that has a number of metabolic effects in the body, including the control of both glucose and fatty acid metabolism. The globular head domain of adiponectin, gAd, has also been shown to increase fatty acid oxidation in skeletal muscle. Within days after birth, a rapid increase in fatty acid oxidation occurs in the heart. We examined whether adiponectin or gAd plays a role in this maturation of cardiac fatty acid oxidation. Plasma adiponectin increased in newborn rabbits following birth: 1.2 +/- 0.3 microg/ml in 1-day-old, 6.8 +/- 1.8 microg/ml in 7-day-old, and 45 +/- 5 microg/ml in 6-week-old rabbits. Because plasma insulin levels decrease and remain low throughout the suckling period, and because this decrease may contribute to the maturation of fatty acid oxidation, we examined the effects of adiponectin and gAd on fatty acid oxidation in isolated perfused 1-day-old rabbit hearts in the presence or absence of 100 microunits/ml insulin. Adiponectin (10 microg/ml) did not alter fatty acid oxidation in the presence of insulin. In the absence of insulin, the addition of recombinant gAd (1.5 microg/ml) increased fatty acid oxidation compared with control (129 +/- 18 versus 66 +/- 11 nmol.g dry weight(-1).min(-1), respectively (p < 0.05). In 7-day-old hearts, where fatty acid oxidation rates were 5-fold higher than 1-day-old hearts, gAd did not alter fatty acid oxidation rates. The increase in fatty acid oxidation in 1-day-old hearts occurred independently of changes in 5'-AMP-activated protein kinase, acetyl-CoA carboxylase, or malonyl-CoA. The effect of gAd on fatty acid oxidation was reversed in the presence of 100 microunits/ml insulin. These results suggest that a decrease in plasma insulin and increase in gAd are involved in the increase of cardiac fatty acid oxidation in the immediate newborn period.     

Metabolism of GnRH in man: influence of estrogens

To clarify the influence of estrogens on the metabolism of gonadotropin-releasing hormone (GnRH), we studied the metabolic clearance rate (MCR) of GnRH (MCRGnRH), and the serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol and testosterone (total and free fraction) in 9 sexually mature men and 7 women under basal conditions and after treatment with the antiestrogen tamoxifen (2 X 10 mg/day p.o.) for 7 days. In women, the medication was started on day 7 +/- 1 of their menstrual cycles. To calculate the MCR, synthetic GnRH was continuously infused (1.53 micrograms/min) and its serum levels were measured by a radioimmunoassay. During tamoxifen treatment we observed a small but significant decrease in the MCR in men (455 +/- 48 to 357 +/- 46 ml/min/1.86 m2), whereas the known cyclic increase in the MCR in women was blunted (1,769 +/- 147 to 1,558 +/- 119 ml/min/1.86 m2). There was a small but significant increase in LH levels in women (8.3 +/- 2.1 to 11.5 +/- 2.5 mU/ml). LH and testosterone levels in men, and FSH and estradiol levels in both sexes did not change significantly. Conclusion: (1) estrogens regulate the MCRGnRH either directly or by changing gonadotropin levels, but the effect is only slight; (2) an enhanced metabolism of GnRH may contribute to the feedback of estrogens on the secretion of gonadotropins, and (3) the sex-specific difference of the MCR is presumably not caused by estrogens.     

Enterocyte viability and mitochondrial function after graded intestinal ischemia and reperfusion in rats

Ischemia/reperfusion of the small intestine can lead to metabolic and structural alterations in the mucosa. Cellular dysfunction occurs when mitochondrial metabolism is compromised, which may ultimately lead to impaired organ function. The aims of this study were to assess the suppression of cellular and mitochondrial oxidative metabolism and involvement of mitochondria in the ischemia/reperfusion injury. The mitochondria were prepared from isolated enterocytes obtained from the small intestine of anesthetized adult rats following different time periods of ischemia and ischemia followed by 5 min reperfusion. Cellular and mitochondrial function were assessed using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay. Ischemia of increasing time periods caused a progressive decrease in cellular and mitochondrial MTT reduction in enterocytes and reperfusion showed further decrease of MTT formazan formation. Inclusion of 1 mM succinate, as respiratory subs trate, showed reversal of suppression of mitochondrial function in 30-60 min ischemia whereas 90 min ischemia or short time period ischemia followed by 5 min reperfusion indicated an irreversible damage to mitochondria. This study indicated that mitochondria are a sensitive target of damage due to oxygen deficiency and possibly due to sudden burst of oxygen free radicals. Mitochondria can withstand short periods of ischemia whereas long duration ischemia or reperfusion results in irreversible damage to mitochondrial function. (Mol Cell Biochem 167: 81-87, 1997)     

Simultaneous determination of coumarin, 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide in human serum and plasma by high-performance liquid chromatography

A HPLC method was developed for the determination of the metabolites of coumarin and 7-hydroxycoumarin in plasma and serum. Separation was based on gradient elution of 7-hydroxycoumarin glucuronide, 7-hydroxycoumarin, coumarin and finally 4-hydroxycoumarin (which is used as an internal standard). Standards, prepared in plasma or serum, and samples were treated with trichloroacetic acid, mixed and centrifuged. The supernatant was removed and analyzed by reversed-phase high-performance liquid chromatography on a C₁₈ column. The limit of detection was 50 ng/ml for 7-hydroxycoumarin and 200 ng/ml for coumarin and 7-hydroxycoumarin glucuronide. The linear range was 0.5–100 μg/ml for each of the analytes. The percentage relative standard deviation about the mean measured concentrations were all below 10%. There was no statistical difference between the standard curves prepared in plasma or serum. The method developed was applied to the determination of each of the three compounds in serum, after the administration of 7-hydroxycoumarin, and in plasma after the administration of coumarin. The concentrations of total 7-hydroxycoumarin in the serum samples were also determined by another HPLC method and the results were compared. There was no statistical difference between the results determined.     

Cholinergic stimulation of the Na+/K+ adenosine triphosphatase as revealed by microphysiometry.

The activation of a wide range of cellular receptors has been detected previously using a novel instrument, the microphysiometer. In this study microphysiometry was used to monitor the basal and cholinergic-stimulated activity of the Na+/K+ adenosine triphosphatase (ATPase) (the Na+/K+ pump) in the human rhabdomyosarcoma cell line TE671. Manipulations of Na+/K+ ATPase activity with ouabain or removal of extracellular K+ revealed that this ion pump was responsible for 8.8 +/- 0.7% of the total cellular energy utilization by those cells as monitored by the production of acid metabolites. Activation of the pump after a period of inhibition transiently increased the acidification rate above baseline, corresponding to increases in intracellular [Na+] ([Na+]i) occurring while the pump was off. The amplitude of this transient was a function of the total [Na+]i excursion in the absence of pump activity, which in turn depended on the duration of pump inhibition and the Na+ influx rate. Manipulations of the mode of energy metabolism in these cells by changes of the carbon substrate and use of metabolic inhibitors revealed that, unlike some other cells studied, the Na+/K+ ATPase in TE671 cells does not depend on any one mode of metabolism for its adenosine triphosphate source. Stimulation of cholinergic receptors in these cells with carbachol activated the Na+/K+ ATPase via an increase in [Na+]i rather than a direct activation of the ATPase.     

Regulation of rat natural killing. II. Inhibition of cytolysis and activation by inhibitors of lipoxygenase: possible role of leukotrienes

Rat splenic natural killer (NK) cell activity against 51Cr-labeled YAC-1 or TMT-081 tumor cells can be augmented by culturing at 37 degrees C for 18 hr. Inhibitors of the lipoxygenase pathway of arachidonic acid metabolism, NDGA, alpha-phenanthroline, quercetin, ETYA, BW755C, esculetin, and timegadine, inhibited this NK activation and also inhibited NK cytotoxicity when added directly to the NK assay. However, there was a partial loss of sensitivity of activated NK cells to suppression by NDGA, BW755C, and esculetin. Indomethacin failed to reverse the inhibition of NK activation caused by NDGA. However, LTB4 and LTC4 (0.01 microgram/ml) were able to reverse the inhibitory effect of NDGA on NK activation. Furthermore, spleen cells cultured for 18 hr synthesized detectable amount of LTC4 in their supernatants. NDGA inhibited the LTC4 synthesis in a dose-dependent manner. These data therefore suggest that leukotrienes are responsible for NK activation, and lipoxygenase activity is essential for NK cytolytic activity.     

Antileishmanial activity of lapachol analogues

The antileishmanial activity of lapachol, isolapachol, and dihydrolapachol, along with soluble derivatives (potassium salt) and acetate was obtained. All the compounds were assayed against metacyclic promastigotes of two different species of Leishmania associated to tegumentar leishmaniasis: L. amazonensis and L. braziliensis. All compounds presented significant activity, being isolapachol acetate the most active against promastigotes, with IC50/24h = 1.6 +/- 0.0 microg/ml and 3.4 +/- 0.5 microg/ml for, respectively, L. amazonensis and L. braziliensis. This compound was also assayed in vivo against L. amazonensis and showed to be active. Its toxicity in vitro was also established, and at concentration similar to the IC50, no toxicity was evidenced. In all experiments, pentamidine isethionate was used as a reference drug. The present results reinforce the potential use of substituted hydroxyquinones and derivatives as promising antileishmanial drugs and suggest a continuing study within this class of compounds.     

Effects of plasma cholesterol lowering agents on hepatobiliary lipid metabolism and cholesterol turnover in the rhesus monkey.

The effects of clofibrate, cholestyramine, and neomycin on hepatobiliary lipid metabolism were studied in adult rhesus monkeys in metabolic steady state with intact but exteriorized enterohepatic circulations. Clofibrate (30 mg/kg, id) had no effect on lipid secretion while cholestyramine (150 mg/kg, id) decreased biliary cholesterol secretion rate from 0.19 +/- 0.03 to 0.13 +/- 0.02 mmol/24 h, p less than 0.05. Neomycin (30 mg/kg, id) decreased bile flow from 216 +/- 10 to 191 +/- 7mL/24 h, p less than 0.05, and tended only to decrease bile salt and phospholipid secretion rates. Cholestyramine decreased cholesterol composition from 1.81 +/- 0.22 to 1.30 +/- 0.22 mol %, p less than 0.05, while clofibrate and neomycin had insignificant effects. Cholestyramine and neomycin decreased bile salt pool size from 1 +/- 0.1 to 0.77 +/- 0.15 and from 1.45 +/- 0.16 to 1.13 +/- 0.21 mmol, p less than 0.05, respectively, while clofibrate had no effect. Bile salt synthetic rate was increased only by cholestyramine, i.e., from 0.63 +/- 0.04 to 1.48 +/- 0.26 mmol/24 h, p less than 0.01. Concomitant cholesterol turnover studies revealed that cholestyramine increased the production rate and excretion of cholesterol in the rapidly miscible cholesterol pool and increased the transfer of cholesterol from slow to rapidly miscible pools. Neomycin, on the other hand, decreased the size of the rapidly miscible pool by decreasing production rate without affecting the size of the slowly miscible pool, while clofibrate had insignificant effects.     

The effect of omega-3 fatty acids on Ca-ATPase in rat cerebral cortex

Neuronal Ca-ATPase has the essential function of keeping intracellular Ca levels in the micromolar range. This is a prerequisite for normal neurotransmission. This study was designed to determine whether Ca-ATPase is a target for docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) action: results show that both these fatty acids are inhibitors of Ca-ATPase activity in synaptosomal membranes isolated from rat cerebral cortex (-65+/-5% at [DHA]=20 microg/ml, -59+/-7% at [EPA]=20 microg/ml). The inhibition caused by EPA, but not that of DHA, could be reversed completely by the addition of calphostin, a protein kinase C blocker. In contrast, DHA could stimulate Ca-ATPase activity (+132+/-5% at [DHA]=30 microg/ml) only in calmodulin-depleted membranes. In addition, Na,K-ATPase (which drives the Na-Ca exchanger) was inhibited by both DHA and EPA, both at 30 microg/ml (-15+/-0.7% and -42+/-1%, respectively). These results suggest a mechanism that explains the dampening effect of omega-3 fatty acids on neuronal activity.     

The rate of P-glycoprotein activation depends on the metabolic state of the cell

P-glycoprotein ATPase activity has been studied almost exclusively by measuring inorganic phosphate release from inside-out cellular vesicles. We have recently proposed a new method based on measurements of the extracellular acidification rate (ECAR) of living cells with a Cytosensor microphysiometer. This method allows for systematic investigation of the various factors influencing P-glycoprotein activation in living cells. Basal metabolic rates or ECARs of different MDR1-transfected cell lines were compared with those of the Mdr1a(-/-)1b(-/-) knockout, MRP1-transfected, and corresponding wild-type cell lines. Basal ECARs of all cells were on the order of 10(7) protons/cell/s, whereby those of genetically modified cells were on average (over all cell lines) slightly lower than those of wild-type cells. The expression level of P-glycoprotein in MDR1-transfected cells had no influence on basal ECARs. Verapamil-induced ECARs were specific for MDR1-transfected cells and increased with the expression level of P-glycoprotein. Moreover, ECARs were dependent on the metabolic state of the cell and were (2.8 +/- 1.2) x 10(6) and (8.0 +/- 1.5) x 10(6) protons/cell/s in glucose-deficient and glucose-fed NIH-MDR-G185 cells, respectively, after verapamil (10 muM) stimulation. The ECARs were practically identical to the rates of lactate extrusion and thus reflect the rates of ATP synthesis via glycolysis. Taking into account the number of P-glycoprotein molecules per cell, the rate of ATP hydrolysis in inside-out vesicles of the same cells was determined as (9.2 +/- 1.5) x 10(6) phosphates/cell/s, in good agreement with the rate of ATP synthesized in glucose-fed cells. The energy required for P-glycoprotein activation relative to the basal metabolic energy was twice as large in glucose-deficient as in glucose-fed cells, suggesting cellular protection by P-glycoprotein even under conditions of starvation.     

The Chinese medicine Bu-Zhong-Yi-Qi-Tang inhibited proliferation of hepatoma cell lines by inducing apoptosis via G0/G1 arrest

Bu-Zhong-Yi-Qi-Tang (BZYQT), a Chinese herbal medicine, inhibited the proliferation of human hepatoma cell lines (Hep3B, HepG2 and HA22T) dose-dependently. The IC50s of BZYQT on the proliferation of Hep3B, HepG2 and HA22T were 432.5+/-31.8 microg/ml, 455.4+/-24.2 microg/ml, and 2284.3+/-77.2 microg/ml respectively on day 3. However, BZYQT did not significantly inhibit the proliferation of normal human hepatocytes (Chang liver, CCL-13) at the concentration under 5,000 microg/ml. Major compounds of BZYQT, including astragaloside IV, ginsenoside Rb1 and Rg1, saikosaponin a and c, and glycyrrhizin, have been identified. To investigate the key inhibitors of BZYQT. Hep3B cells were treated with BZYQT, individual major compounds of BZYQT, and mixture of major compounds in the same ratio as present in BZYQT. Significant inhibition of proliferation was detected in BZYQT and its major compounds mixture in a comparable level. Not any individual major compound examined could suppress the proliferation of Hep3B cells. This data indicated that there could be synergistic or additive effects of the ingredients in BZYQT. BrdU incorporation, cell cycle analysis and DNA fragmentation assay revealed that BZYQT suppressed the proliferation of hepatoma cells via G0/G1 cell cycle arrest and inhibition of DNA synthesis followed by apoptosis.     

Antioxidant potential of n-butanol fraction from extract of Jasminum mesnyi Hance leaves

Methanolic extract of Jasminum mesnyi Hance leaves having antidiabetic activity was subjected to fractionation to obtain antioxidant and antihyperglycemic rich fraction. Different concentrations of ethyl acetate and n-butanol fractions were subjected to antioxidant assay by DPPH method, nitric oxide scavenging activity and reducing power assay. The fractions showed dose dependent free radical scavenging property in all the models. IC50 values for ethyl acetate and n-butanol fractions were 153.45 +/- 6.65 and 6.22 +/- 0.25 microg/ml, respectively, as compared to L-ascorbic acid and rutin (as standards; IC50 values 6.54 +/- 0.24 and 5.43 +/- 0.21 microg/ml, respectively) in DPPH model. In nitric oxide scavenging activity, IC50 values were 141.54 +/- 9.95 microg/ml, 35.12 +/- 1.58 microg/ml, 21.06 +/- 0.95 microg/ml and 29.93 +/- 0.32 microg/ml for ethyl acetate, n-butanol fractions, L-ascorbic acid and rutin, respectively. n-Butanol fraction showed a good reducing potential and better free radical scavenging activity as compared to ethyl acetate fraction. Potent antioxidant n-butanol fraction showed better oral glucose tolerance test (antihyperglycemic) at par with metformin (standard drug), n-Butanol fraction contained secoiridoid glycosides which might be responsible for both antioxidant and antihyperglycemic activity.     

Evaluation of enzyme histochemical observations for metabolic studies. A combined histochemical and biochemical investigation of experimentally induced skeletal muscle-changes

Summary The reliability of enzyme histochemical observations for metabolic studies on skeletal muscle tissue was investigated with a combined histochemical and biochemical study. Specimens of musculus soleus with a predominantly aerobic metabolism and of musculus flexor digitorum longus with a predominantly anaerobic metabolism of rabbits in which both muscles were surgically cross-reinnervated or auto-reinnervated were used. For the histochemical investigation activities and localisations of succinate dehydrogenase, l-glycerol-3-phosphate: acceptor oxidoreductase, nicotinamide adenine dinucleotide: tetrazolium oxidoreductase and of -glucan phosphorylase were examined. For the biochemical investigation maximal activity of phosphofructokinase, the rate limiting enzyme for the regulation of the glycolysis was measured. In addition the activities of succinate dehydrogenase and l-glycerol-3-phosphate: acceptor oxidoreductase to characterize the aerobic metabolism and the key role in gearing energy requirements to glycolysis respectively were biochemically determined. For further information about metabolic aspects the isoenzyme ratio of lactate dehydrogenase was established. In the present paper the histochemical findings are reported and discussed.Part of this study was taken from the Ph. D. thesis of A. C. Jöbsis (1971).     

Cyclooxygenase enzyme inhibitory compounds with antioxidant activities from Piper methysticum (kava kava) roots.

Cyclooxygenase enzyme inhibitory assay-guided purification of ethyl acetate extract of Piper methysticum (kava kava) roots yielded six biologically active compounds (1-7), which were purified using MPLC, preparative TLC and HPLC methods. These compounds were also evaluated for antioxidant activities. Dihydrokawain (1) and yangonin (6) showed the highest COX-I and COX-II inhibitory activities at 100 microg/ml, respectively. The lipid oxidation assay did not reveal antioxidant activities for demethoxyangonin (2), dihydrokawain (1), kawain (4), dihydromethysticin (5) or methysticin (7) at 50 microg/ml. The antioxidant activities of flavokawain A (3) and yangonin (6) could not be tested in the lipid oxidation assay due to solubility problems. However, yangonin and methysticin showed moderate antioxidant activities in the free radical scavenging assay at 2.5 mg/ml.     

Cyto- and genotoxicity of ultrafine TiO2 particles in cultured human lymphoblastoid cells

Titanium dioxide is frequently used in the production of paints, paper, plastics, welding rod-coating material, and cosmetics, because of its low toxicity. However, recent studies have shown that nano-sized or ultrafine TiO(2) (UF-TiO(2)) (<100 nm in diameter) can generate pulmonary fibrosis and lung tumor in rats. Cytotoxicity induced by UF-TiO(2) in rat lung alveolar macrophages was also observed. This generates great concern about the possible adverse effects of UF-TiO(2) for humans. The cytotoxicity and genotoxicity of UF-TiO(2) were investigated using the methyl tetrazolium cytotoxicity (MTT) assay, the population growth assay, the apoptosis assay by flow cytometry, the cytokinesis block micronucleus (CBMN) assay, the comet assay, and the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation assay. WIL2-NS cells were incubated for 6, 24 and 48 h with 0, 26, 65 and 130 microg/ml UF-TiO(2). Significant decreases in viability were seen in the MTT assay at higher doses; for example, 61, 7 and 2% relative viability at 130 microg/ml for 6, 24 and 48-h exposure (P<0.01). A dose-dependent relationship was observed, while a time-dependent relationship was seen only at the highest dose (130 microg/ml) after exposure for 24 and 48 h. Treatment with 130 microg/ml UF-TiO(2) induced approximately 2.5-fold increases in the frequency of micronucleated binucleated cells (P<0.01). In addition, a significant reduction in the cytokinesis block proliferation index was observed by the CBMN assay (P<0.05). In the comet assay, treatment with 65 microg/ml UF-TiO(2) induced approximately 5-fold increases in olive tail moment (P<0.05). In the HPRT mutation assay, treatment with 130 microg/ml UF-TiO(2) induced approximately 2.5-fold increases in the mutation frequency (P<0.05). The results of this study indicate that UF-TiO(2) can cause genotoxicity and cytotoxicity in cultured human cells.