DNA strand break induction and enhanced cytotoxicity of propyl gallate in the presence of copper(II)

The antioxidant propyl gallate (PG) induced single strand breaks in PM2 DNA at concentrations higher than 0.25 microM when it was combined with copper concentrations at 5 microM and above. In combination with 100 microM CuCl2, extensive double strand breakage was also observed. Neither PG alone nor CuCl2 showed any strand breaking properties. DNA strand breakage was inhibited by addition of catalase or the Cu(I) chelator neocuproine, indicating the involvement of H2O2 and a Cu(II)/Cu(I) redox cycle in the DNA damage. DNA damage of PG/Cu(II) was also observed in human fibroblasts. Using the alkaline elution technique concentrations of 0.15-0.5 mM PG induced DNA strand breaks in combination with 2.5 mM CuCl2, while the single substances did not show any effect. At these concentrations cell viability measured by the MTT assay was not reduced by more than 10%; however, cell growth was inhibited by PG in combination with Cu(II). This growth inhibition was apparently due to the DNA damage incurred by PG/Cu(II). The synergistic interaction between PG and Cu(II) is probably caused by a redox reaction between both compounds, whereby reactive species such as ROS are formed, which are responsible for the observed genotoxic and cytotoxic effects. Our results demonstrate that the antioxidative and cytoprotective properties of propyl gallate may change to prooxidative, cytotoxic and genotoxic properties in the presence of Cu(II).     

Interaction of histone H1 with cis-platinum modified DNA

Cis-diamminedichloroplatinum(II) (cis-DDP) is known as an effective anticancer drug. Its therapeutic effect is supposed to be a consequence of the covalent binding to DNA. A number of cellular proteins were found to bind selectively to DNA modified by cis-DDP (but not by its isomer trans-DDP). Here we present our observations on interaction of the linker histone H1 with cis- and trans-DDP modified DNA fragments. The results afford new experimental information about the preferential binding of histone H1 to cis-DDP-distorted DNAs versus trans-DDP modified ones.     

Stimulatory effect of regucalcin on ATP-dependent calcium transport in rat liver plasma membranes

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytoplasm, on ATP-dependent calcium transport in the plasma membrane vesicles of rat liver was investigated. (Ca(2+)- Mg2+)-ATPase activity in the liver plasma membranes was significantly increased by the presence of regucalcin (0.1-0.5 microM) in the enzyme reaction mixture. This increase was completely inhibited by the presence of sulfhydryl group modifying reagent Nethylmaleimide (5.0 mM NEM) or digitonin (0.04%), which can solubilize the membranous lipids. When ATP-dependent calcium uptake by liver plasma membrane vesicles was measured by using 45CaCl2, the presence of regucalcin (0.1-0.5 microM) in the reaction mixture caused a significant increase in the 45Ca2+ uptake. This increase was about 2-fold with 0.5 microM regucalcin addition. An appreciable increase was seen by 5 min incubation with regucalcin addition. The regucalcin-enhanced ATP-dependent 45Ca2+ uptake by the plasma membrane vesicles was completely inhibited by the presence of NEM (5.0 mM) or digitonin (0.04%). These results demonstrate that regucalcin activates (Ca(2+)-Mg2+)-ATPase in the liver plasma membranes and that it can stimulate ATP-dependent calcium transport across the plasma membranes.     

Inhibitory effect of 9-(2-phosphonylmethoxyethyl)-adenine (PMEA) on human and duck hepatitis B virus infection

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) was evaluated for its inhibitory effect on hepadnavirus replication in three different cell systems, i.e., human hepatoma cell lines HepG2 2.2.15 and HB611 (transfected with human hepatitis B virus (HBV)) and primary cultures of duck hepatocytes infected with duck hepatitis B virus (DHBV). PMEA inhibited HBV release from HepG2 2.2.15 cells and HB611 cells at a 50% inhibitory concentration (IC50) of 0.7 and 1.2 microM, respectively. Intracellular viral DNA synthesis was inhibited at concentrations equivalent to those required to inhibit virus release from the cells. DHBV secretion from duck hepatocytes was inhibited by PMEA at an IC50 of 0.2 microM. HBsAg secretion was inhibited by PMEA in a concentration-dependent manner in HB611 cells and DHBV-infected duck hepatocytes but not HepG2 2.2.15 cells. The 50% cytotoxic concentration, as measured by inhibition of [3H-methyl]deoxythymidine incorporation was 150 microM for the two human hepatoma cell lines and 40 microM for the duck hepatocyte cultures. In a pilot experiment PMEA was found to reduce the amounts of DHBV DNA in the serum of Pekin ducks.     

Interaction of the renin-angiotensin system, bradykinin and sympathetic nerves with cholinergic transmission in the rat isolated trachea

1. The present study was undertaken to investigate the interaction of the renin-angiotensin system (RAS), bradykinin and the sympathetic nervous system with cholinergic transmission in the rat airways. Experiments were performed on epithelium-intact and epithelium-denuded preparations of rat isolated trachea which had been incubated with [3H]-choline to incorporate [3H]-acetylcholine into the cholinergic transmitter stores. Tracheal preparations were subjected to electrical field stimulation (trains of 1 ms pulses, 5 Hz, 15 V) and the stimulation-induced (S-I) efflux taken as an index of transmitter acetylcholine release. 2. In both epithelium-intact and epithelium-denuded tracheal preparations, the alpha 2-adrenoceptor agonist UK14304 (0.1 and 1 microM) inhibited the S-I efflux, in a concentration-dependent manner. The inhibition of S-I efflux produced by UK14304 (1 microM) was antagonized by the selective alpha 2-adrenoceptor antagonist idazoxan (0.3 microM). Idazoxan (0.3 microM) alone had no effect on the S-I efflux. 3. Angiotensin II (0.1 and 1 microM) was without effect on the S-I efflux in either epithelium-intact or epithelium-denuded tracheal preparations. When angiotensin-converting enzyme was inhibited by perindoprilat (10 microM), angiotensin II (1 microM) was also without effect on the S-I efflux. Similarly, in the presence of idazoxan (0.3 microM), to block prejunctional alpha 2-adrenoceptors, angiotensin II (0.1 and 1 microM) did not alter the S-I efflux. When added alone, perindoprilat (10 microM) did not alter the S-I efflux. 4. In epithelium-denuded preparations, bradykinin (0.01-1 microM) inhibited the S-I efflux. In epithelium-intact preparations, there was also a tendency for bradykinin (0.1 and 1 microM) to inhibit the S-I efflux but this was not statistically significant. However, when angiotensin-converting enzyme and neutral endopeptidase were inhibited by perindoprilat (10 microM) and phosphoramidon (1 microM), respectively, bradykinin (1 microM) significantly inhibited the S-I efflux in epithelium-intact preparations as well as in epithelium-denuded preparations. The inhibition of the S-I efflux produced by bradykinin, in the combined presence of perindoprilat (10 microM) and phosphoramidon (1 microM), was unaffected by the additional presence of the cyclo-oxygenase inhibitor indomethacin (10 microM) and/or the nitric oxide synthase inhibitor NG-nitro-L-arginine (100 microM), in either epithelium-intact or epithelium-denuded preparations. 5. In conclusion, the findings of the present study suggest that airway parasympathetic nerves are endowed with alpha 2-adrenoceptors which subserve inhibition of transmitter acetylcholine release. Under the present conditions, however, transmitter acetylcholine release is not subject to transneuronal modulation by noradrenaline released from adjacent sympathetic nerves in the airways. Moreover, angiotensin II and perindoprilat do not appear to modulate acetylcholine release from parasympathetic nerves of the airways. In contrast, bradykinin inhibits acetylcholine release from airway parasympathetic nerves but this action of bradykinin is limited by the activity of epithelial angiotensin-converting enzyme and/or neutral endopeptidase. The inhibitory action of bradykinin on cholinergic transmission in the airways does not appear to involve the liberation of prostaglandins or nitric oxide.     

DNA strand scission by polycyclic aromatic hydrocarbon o-quinones: role of reactive oxygen species, Cu(II)/Cu(I) redox cycling, and o-semiquinone anion radicals,

In previous studies, benzo[a]pyrene-7,8-dione (BPQ), a polycyclic aromatic hydrocarbon (PAH) o-quinone, was found to be 200-fold more potent as a nuclease than (+/-)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene, a suspect human carcinogen. The mechanism of strand scission mediated by naphthalene-1,2-dione (NPQ) and BPQ was further characterized using either phiX174 DNA or poly(dG).poly(dC) as the target DNA. Strand scission was extensive, dependent on the concentration of o-quinone (0-10 microM), and required the presence of NADPH (1 mM) and CuCl2 (10 microM). The production of reactive species, i.e., superoxide anion radical, o-semiquinone anion (SQ) radical, hydrogen peroxide (H2O2), hydroxyl radical (OH.), and Cu(I), was measured in the incubation mixtures. The formation of SQ radicals was measured by EPR spectroscopy under anaerobic conditions in the presence of NADPH. A Cu(II)/Cu(I) redox cycle was found to be critical for DNA cleavage. No strand scission occurred in the absence of Cu(II) or when Cu(I) was substituted, yet Cu(I) was required for OH* production. Both DNA strand scisson and OH. formation were decreased to an equal extent, albeit not completely, by the inclusion of OH. scavengers (mannitol, soduim benzoate, and formic acid) or Cu(I) chelators (bathocuproine and neocuproine). In contrast, although the SQ radical signals of NPQ and BPQ were quenched by DNA, no strand scission was observed. When calf thymus DNA was treated with PAH o-quinones, malondialdehyde (MDA) was released by acid hydrolysis. The formation of MDA was inhibited by OH. scavengers suggesting that OH* cleaved the 2'-deoxyribose moiety in the DNA to produce base propenals. These studies indicate that for PAH o-quinones to act as nucleases, NADPH, Cu(II), Cu(I), H2O2, and OH*, were necessary and that the primary species responsible for DNA fragmentation was OH., generated by a Cu(I)-catalyzed Fenton reaction. The genotoxicity of PAH o-quinones may play a role in the carcinogenicity and mutagenicity of the parent hydrocarbons.     

Effect of silibinin on oxidative damage of blood constituents

Silibinin (SDH) is a flavonoid with ascertained hepatoprotective effects, which have been partially attributed to its antioxidant properties. Oxidation of blood constituents could have a role in atherogenesis and interfere with the rheologic properties of the blood. In this study we investigated, whether SDH could protect some blood constituents against oxidative modification. In human plasma we measured TBARS and fluorescence generation as indicators of copper or azobis amidinopropane hydrochloride (AAPH) at 760 mm Hg PO2-induced lipid peroxidation. SDH at 50 microM inhibited copper-induced TBARS formation by 25% and fluorescence by 47%. SDH also inhibited AAPH-induced lipid peroxidation, but at 175 microM concentration only. Oxidative modification of albumine was evaluated by fluorescence generation. SDH at 50 microM inhibited copper/hydrogen peroxide fluorescence generation by 54% and at 2.5 microM it inhibited EDTA-Fe (II)/hydrogen peroxide fluorescence generation by 31%. The protection of albumin by SDH was confirmed by SDS-PAGE electrophoresis. Copper-induced red-cell lipid peroxidation was evaluated by TBARS formation. SDH at 250 microM inhibited copper-induced lipid peroxidation and hemolysis by 45% and 94%, respectively. SDH also inhibited hemolysis in red-cell suspensions exposed to hydrogen peroxide, but not lipid peroxidation. Our results show that SDH may protect blood constituents from oxidative damage.     

Role of caspases (ICE/CED 3 proteases) in DNA damage and cell death in response to a mitochondrial inhibitor, antimycin A

Caspases (ICE/ Ced3 proteases) are a closely related family of cysteine proteases that play a key role in apoptotic cell death. We examined the role of caspases in DNA damage and cell death in response to the mitochondrial inhibitor, antimycin A. LLC-PK1 cells contain caspase activity that was markedly inhibited by cleavage site-based peptide inhibitors of caspases but not by inhibitors of serine, cysteine, aspartate or metalloproteinases. The caspase activity increased within five minutes of exposure to antimycin A, preceding any evidence of DNA damage and cell death. The specific caspase inhibitors. Ac-Tyr-Val-Ala-Asp-aldehyde (inhibitor I) and Ac-Asp-Glu-Val-Asp-aldehyde (inhibitor II) prevented, in a dose dependent manner, antimycin A-induced DNA strand breaks as determined by DNA unwinding assay (residual double stranded DNA in control, 94 +/- 2%; antimycin A alone, 48 +/- 3%; antimycin A + inhibitor I at 50 microM, 93 +/- 2%; antimycin A + inhibitor II at 50 microM, 89 +/- 5%; N = 3 to 4, P < 0.001). These inhibitors also prevented antimycin A-induced DNA fragmentation as determined by agarose gel electrophoresis and by in situ labeling of cell nuclei by the terminal deoxynucleotidyl transferase (TdT) nick end labeling (TUNEL) method. The caspase inhibitors markedly prevented antimycin A-induced cell death in a dose-dependent manner as measured by trypan blue exclusion (control 6 +/- 1%, antimycin A alone 40 +/- 1%, antimycin A + inhibitor I at 50 microM 16 +/- 1%, antimycin A + inhibitor II at 50 microM 16 +/- 1%; N = 4 to 7, P < 0.001). These data indicate that the caspase family of enzymes play an important role in DNA damage and cell death in response to the mitochondrial inhibitor, antimycin A.     

Direct effects of diazoxide on mitochondria in pancreatic B-cells and on isolated liver mitochondria

1. The direct effects of diazoxide on mitochondrial membrane potential, Ca2+ transport, oxygen consumption and ATP generation were investigated in mouse pancreatic B-cells and rat liver mitochondria. 2. Diazoxide, at concentrations commonly used to open adenosine 5'-triphosphate (ATP)-dependent K+-channels (K(ATP) channels) in pancreatic B-cells (100 to 1000 microM), decreased mitochondrial membrane potential in mouse intact perifused B-cells, as evidenced by an increase of rhodamine 123 fluorescence. This reversible decrease of membrane potential occurred at non-stimulating (5 mM) and stimulating (20 mM) glucose concentrations. 3. A decrease of mitochondrial membrane potential in perifused B-cells was also caused by pinacidil, but no effect could be seen with levcromakalim (500 microM each). 4. Measurements by a tetraphenylphosphonium-sensitive electrode of the membrane potential of rat isolated liver mitochondria confirmed that diazoxide decreased mitochondrial membrane potential by a direct action. Pretreatment with glibenclamide (2 microM) did not antagonize the effects of diazoxide. 5. In Fura 2-loaded B-cells perifused with the Ca2+ channel blocker, D 600, a moderate, reversible increase of intracellular Ca2+ concentration could be seen in response to 500 microM diazoxide. This intracellular Ca2+ mobilization may be due to mitochondrial Ca2+ release, since the reduction of membrane potential of isolated liver mitochondria by diazoxide was accompanied by an accelerated release of Ca2+ stored in the mitochondria. 6. In the presence of 500 microM diazoxide, ATP content of pancreatic islets incubated in 20 mM glucose for 30 min was significantly decreased by 29%. However, insulin secretion from mouse perifused islets induced by 40 mM K+ in the presence of 10 mM glucose was not inhibited by 500 microM diazoxide, suggesting that the energy-dependent processes of insulin secretion distal to Ca2+ influx were not affected by diazoxide at this concentration. 7. The effects of diazoxide on oxygen consumption and ATP production of liver mitochondria varied depending on the respiratory substrates (5 mM succinate, 10 mM alpha-ketoisocaproic acid, 2 mM tetramethyl phenylenediamine plus 5 mM ascorbic acid), indicating an inhibition of respiratory chain complex II. Pinacidil, but not levcromakalim, inhibited alpha-ketoisocaproic acid-fuelled ATP production. 8. In conclusion, diazoxide directly affects mitochondrial energy metabolism, which may be of relevance for stimulus-secretion coupling in pancreatic B-cells.     

The electrophoretic separation of curved cisplatin-modified DNA fragments on polyacrylamide gels is dependent on the voltage gradient

Polyacrylamide gel electrophoresis has been widely used to study DNA fragments containing sequence-dependent curvature. The anomalous electrophoretic behavior of curved DNA fragments on such gels allows their separation from straight fragments of the same length. Here we demonstrate that polyacrylamide gels can be successfully used to resolve DNA fragments modified at a single site by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) from their unmodified counterparts. However, the resolution strongly depends on the voltage gradient, being completely lost when it drops below a certain threshold level. The parameters of the electric field do not affect separation of 'normal' DNA fragments of comparable length.     

Site-specific DNA damage induced by NADH in the presence of copper(II): role of active oxygen species

Oxidative DNA damage by NAD(P)H in the presence of metal ions has been characterized by using 32P 5' end-labeled DNA fragments obtained from human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. NADH, as well as other endogenous reductants, induced DNA damage in the presence of Cu(II). The order of inducing effect on Cu(II)-dependent DNA damage was ascorbate > reduced glutathione (GSH) > NADH > NADPH. Although NADH caused no or little DNA damage in the presence of Fe(III)-EDTA, the addition of H2O2 induced the DNA damage. The Cu(II)-mediated DNA damage induced by NADH was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator; but not by scavengers of hydroxyl free radical (.OH), suggesting the involvement of active species derived from hydrogen peroxide (H2O2) and Cu(I) rather than .OH. The predominant cleavage sites were thymine residues located 5' and/or 3' to guanine. The cleavage pattern was similar to that induced by Cu(II) plus GSH, Cu(II) plus ascorbate, or Cu(I) plus H2O2. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by NADH increased with its concentration in the presence of Cu(II). UV-visible spectroscopy indicated the facilitation of reduction of Cu(II) by NADH under some conditions. ESR spin-trapping experiments and mass spectrometry showed that the carbon-centered radical was formed during the reaction of NADH with Cu(II). These results suggest that optimal molar ratios of DNA/metal ion yield copper with a high redox potential which catalyzes NADH autoxidation to NAD. being further oxidized to NAD+ with generation of superoxide radical and that H2O2 reacts with Cu(I) to form active oxygen species such as copper(I)-peroxide complex causing DNA damage.     

High selectivity for inhibition of phosphodiesterase III and positive inotropic effects of MCI-154 in guinea pig myocardium

MCI-154 (0.3-100 microM) exerted a concentration-dependent positive inotropic effect in isolated guinea pig papillary muscles (EC50 0.8 microM). The efficacy of MCI-154 (253% of predrug value) was 1.7-fold higher than that of saterinone but comparable to that of milrinone. Carbachol markedly reduced the increase in force of contraction (FOC) of MCI-154. In intact contracting papillary muscles, the positive inotropic effect was accompanied by an increase in cyclic AMP content to 0.78 +/- 0.09 pmol/mg wet weight (n = 10), corresponding to 150% of the basal value (0.51 +/- 0.05 pmol/mg wet weight, n = 21) in the presence of submaximal cyclic AMP phosphodiesterase (PDE) isoenzyme III inhibiting concentrations of MCI-154 (30 microM). MCI-154 (1-1,000 microM) concentration-dependently inhibited the activity of PDE III from homogenates of guinea pig myocardium. The IC50 was 3.8 microM. PDE I, II, and IV were not significantly affected up to 100 microM (PDE I and IV) and up to 1,000 microM (PDE II). In comparison, milrinone and saterinone were PDE III/IV-selective PDE inhibitors. Rolipram inhibited PDE IV only. IBMX and theophylline were nonselective PDE inhibitors. MCI-154 had only a marginal positive chronotropic effect. The frequency of spontaneously beating right auricles from guinea pig heart was increased by 8.7% at most (n = 5). MCI-154 increased Ca2+ sensitivity in chemically skinned porcine ventricular muscle fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effects of formoterol on platelet-activating factor induced eosinophil chemotaxis and degranulation

A new long-acting beta 2-agonist, formoterol, has been reported to have a greater efficacy and duration of action in asthmatic patients as compared to conventional beta 2-agonists. We recently demonstrated that formoterol inhibited antigen-induced late asthmatic response (LAR) and accompanying airway eosinophilia in guinea pigs. In this study, we investigated the direct effect of formoterol in vitro on human eosinophil function, focusing on platelet-activating factor (PAF)-induced eosinophil chemotaxis and eosinophil cationic protein (ECP) release. Purified normodense eosinophils were separated by discontinuous gradient from 12 mild asthmatic patients. Formoterol in concentrations of 1-100 microM significantly inhibited PAF-induced eosinophil chemotaxis in a dose-dependent manner with a concentration of drug required to produce 50% inhibition (IC50) of 10.16 microM; % inhibition: 22.9 +/- 13.0% (1 microM), 51.6 +/- 12.7% (10 microM), 75.0 +/- 11.3% (100 microM). When formyl-methionyl-leucyl-phenylamine (FMLP) was used as a chemoattractant, a similar inhibition of eosinophil chemotaxis by formoterol was observed; % inhibition: 13.1 +/- 5.0% (1 microM). 47.7 +/- 7.6% (10 microM), 65.5 +/- 16.5% (100 microM). A conventional beta 2-agonist, salbutamol, at doses to 100 microM did not show any inhibitory effects on PAF-induced eosinophil chemotaxis. Formoterol in concentrations of 1-100 microM also significantly inhibited PAF-induced ECP release from eosinophils; % inhibition: 21.7 +/- 9.0% (1 microM), 39.3 +/- 7.4% (10 microM), 39.6 +/- 8.4% (100 microM). In the presence of phosphodiesterase inhibitors, theophylline or isobutylmethyl xanthine (IBMX), the inhibition by formoterol on PAF-induced ECP release was enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of mammalian DNA topoisomerase I-mediated DNA cleavage and DNA winding by bulgarein

Bulgarein, a fungal metabolite, induced mammalian topoisomerase I-mediated DNA cleavage in vitro. The cleavage activity of bulgarein was comparable to that of camptothecin at a drug concentration range of 0.025-approximately 5 microM. The DNA cleavage induced by bulgarein was suppressed at concentrations above 12.5 microM. Treatment of a reaction mixture containing bulgarein and topoisomerase I with elevated temperature (65 degrees C) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I-mediated DNA cleavage induced by bulgarein is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex." Intensity of cleaved DNA fragments induced by bulgarein with topoisomerase I was different from that induced by camptothecin. Bulgarein inhibited catalytic activities of both topoisomerase I and topoisomerase II. The changes in absorption spectra of bulgarein in the visible region observed upon addition of increasing amounts of calf thymus DNA indicate that bulgarein interacts with DNA. DNA (un)winding assay by two-dimensional gel electrophoresis showed that bulgarein induced the winding of DNA in the opposite direction to that of an intercalator so that positively supercoiled molecules are produced. Thus, bulgarein represents a new class of drugs which stabilizes the cleavable complex of topoisomerase I and alters the structure of DNA in a manner leading to a tightening of the helical twist.     

Bathing premature infants: physiological and behavioral consequences

Phytoestrogen effects on estrogen action and tyrosine kinase activity have been proposed to contribute to cancer prevention. To study these mechanisms, a number of phytoestrogens and related compounds were evaluated for their effects on DNA synthesis (estimated by thymidine incorporation analysis) in estrogen-dependent MCF-7 cells in the presence of estradiol (E2), tamoxifen, insulin, or epidermal growth factor. We observed that 1) at 0.01-10 microM, genistein and coumestrol enhanced E2-induced DNA synthesis, as did 10 microM enterolactone. Chrysin at 1.0-10 microM and 10 microM luteolin or apigenin inhibited E2-induced DNA synthesis, as did all compounds at > 10 microM, 2) tamoxifen enhanced genistein-induced DNA synthesis but inhibited DNA synthesis induced by all other compounds, and 3) genistein enhanced insulin- and epidermal growth factor-induced DNA synthesis at 0.1-1.0 and 0.1-10 microM, respectively. At higher concentrations, inhibition was observed. Similar effects were seen with coumestrol. In conclusion, the effects of phytoestrogens in the presence of E2 or growth factors are concentration dependent and variable. At low concentrations, genistein and coumestrol significantly enhanced E2-induced and tyrosine kinase-mediated DNA synthesis; at high concentrations, inhibition was observed. Differing effects were observed with the other compounds. The variable effects of phytoestrogens on DNA synthesis must be considered when their roles in cancer prevention or treatment are evaluated.     

Acetyl-11-keto-beta-boswellic acid induces apoptosis in HL-60 and CCRF-CEM cells and inhibits topoisomerase I

Antiproliferative action of different pentacyclic triterpenes has repeatedly been reported, and some lipoxygenase inhibitors have been shown to induce cell death in various cell systems. Acetyl-11-keto-beta-boswellic acid (AKBA) is a pentacyclic triterpene that inhibits 5-lipoxygenase in a selective, enzymedirected, nonredox, and noncompetitive manner. To investigate a possible effect of AKBA on leukemic cell growth, proliferation of HL-60 and CCRF-CEM cells was assayed in the presence of AKBA and a structural analog without effect on 5-lipoxygenase, amyrin. Cell counts and [3H]thymidine incorporation were significantly reduced in a dose-dependent manner in the presence of AKBA (IC50 = 30 microM) but not amyrin. An additive effect of AKBA with the crosslinking of the CD95 receptor was also observed. Flow cytometric analysis of propidium iodide-stained cells indicated that the cells underwent apoptosis. This was confirmed by flow cytometric detection of sub-G1 peaks in AKBA-treated cells and by DNA laddering. However, because HL-60 and CCRF-CEM do not express 5-lipoxygenase mRNA constitutively, a mechanism distinct from inhibition of 5-lipoxygenase must account for the effect of AKBA. In a DNA relaxation assay with phiX174RF DNA, AKBA inhibited topoisomerase I from calf thymus at concentrations of >/=10 microM. A semiquantitative cDNA polymerase chain reaction approach was used to estimate the relative level of expression of topoisomerases in both cell lines. The data suggest that induction of apoptosis in HL-60 and CCRF-CEM by AKBA may be due to inhibition of topoisomerase I in these cells.     

Cardiac manifestations of polymyositis. Apropos of 2 Senegalese cases]

The relaxations mediated by the activation of 5-HT receptors in the guinea pig proximal colon were investigated. Longitudinal strips were cut from the colon segment and placed into the bath. In the presence of atropine (0.2 microM), the relaxations were evoked by adding increasing concentrations of 5-HT (1-100 microM). Noncumulative concentration-response curves were established in the absence and presence of either 5-HT or nitric oxide synthase (NOS) antagonists. Selective 5-HT3 antagonists tropisetron (10 and 100 nM) and ondansetron (1 microM) inhibited the relaxations and shifted the concentration-response curves to the right. Similar effects were observed in the presence of the NOS inhibitor N(G)-nitro-L-arginine (3.2, 10, 32 microM) and partly reversed with L-arginine (100, 320 microM). N(G)-nitro-D-arginine, serving as a negative control, was ineffective. The relaxations were further inhibited in the presence of the soluble guanylate cyclase blocker methylene blue (10 microM) or NO scavenger hemoglobin (32 microM). These results suggest that the 5-HT3 receptor plays a role in neurogenic relaxations of guinea pig proximal colon, which are at least partly mediated via release of NO from nerve endings.