Enhancing effects of bovine serum albumin on cell injury in vitro induced with Fe-NTA

Direct effects of ferric nitrilotriacetate (Fe-NTA) on normal rat liver epithelial cells (RL34) in serum-free culture were studied. More than 10 micrograms/ml iron of Fe-NTA was cytotoxic and the cytotoxicity was prevented by adding apotransferrin into culture medium. Also, bovine serum albumin (BSA), which is known to bind Fe-NTA, was found to promote the cytotoxicity, while fatty acids-free BSA (F-BSA) prevented it. This result indicates that fatty acids rather than albumin promote Fe-NTA cytotoxicity.     

Rapid in vitro transformation system for liver epithelial cells by iron chelate,Fe-NTA

We have previously found toxic effects of iron chelate, Fe-NTA on cultured normal rat liver epithelial cells (RL34). In the present study, when RL34 cells were exposed to 50 g/ml iron of Fe-NTA for 15 days, besides the expected cytolytic effects in most cells, the appearance of resistant cells was observed. The resistant cells showed drastic morphological transformation, grew in soft agar, and induced hepatocellular carcinomas when transplanted into syngeneic newborn rats in a short period of time. Since DNA instability in the transformed cells was ascertained by differential AO staining, it is suggested that DNA damage by Fe-NTA is of a critical importance for extremely rapid neoplastic transformation of normal epithelial cells.Abbreviations AO acridine orange - DME medium Dulbecco's modified Eagle's medium - FCS fetal calf serum - Fe-HEDTA ferric-N-(2-hydroxyethyl)ethylenediaminetriacetic acid - Fe-NTA ferric-nitrilotriacetate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PBS Dulbecco's phosphate-buffered saline     

Iron-overload induces oxidative DNA damage in the human colon carcinoma cell line HT29 clone 19A

Dietary iron may contribute to colon cancer risk via production of reactive oxygen species (ROS). The aim of the study was to determine whether physiological ferric/ferrous iron induces oxidative DNA damage in human colon cells. Therefore, differentiated human colon tumour cells (HT29 clone 19A) were incubated with ferric-nitrilotriacetate (Fe-NTA) or with haemoglobin and DNA breaks and oxidised bases were determined by microgelelectrophoresis. The effects of Fe-NTA were measured with additional H(2)O(2) (75microM) and quercetin (25-100microM) treatment. Analytic detection of iron in cell cultures, treated with 250microM Fe-NTA for 15 min to 24h, showed that 48.02+/-5.14 to 68.31+/-2.11% were rapidly absorbed and then detectable in the cellular fraction. Fe-NTA (250-1000microM) induced DNA breaks and oxidised bases, which were enhanced by subsequent H(2)O(2) exposure. Simultaneous incubation of HT29 clone 19A cells with Fe-NTA and H(2)O(2) for 15 min, 37 degrees C did not change the effect of H(2)O(2) alone. The impact of Fe-NTA and H(2)O(2)-induced oxidative damage is reduced by the antioxidant quercetin (75-67% of H(2)O(2)-control). Haemoglobin was as effective as Fe-NTA in inducing DNA damage. From these results we can conclude that iron is taken up by human colon cells and participates in the induction of oxidative DNA damage. Thus, iron or its capacity to catalyse ROS-formation, is an important colon cancer risk factor. Inhibition of damage by quercetin reflects the potential of antioxidative compounds to influence this risk factor. Quantitative data on the genotoxic impact of ferrous iron (e.g. from red meat) relative to the concentrations of antioxidants (from plant foods) in the gut are now needed to determine the optimal balance of food intake that will reduce exposure to this type of colon cancer risk factor.     

Enzymatic inhibition of lysine transport across the small intestine in vivo

1. Trypsin, at different concentrations, significantly inhibited lysine absorption (P less than 0.05) in a dose-dependent pattern. 2. Maximum inhibition equivalent to 35% below control value was reached with 10 micrograms/ml (100 BAEE units) trypsin with a non-reversible inhibitory effect. 3. Chymotrypsin at 10 micrograms/ml produced a significant decrease (P less than 0.05) of lysine absorption although it did not exceed 5%. Perfusion of both enzymes did not show an additive inhibitory effect. 4. Lysine absorption showed a 39% decrease with 10 micrograms/ml trypsin and 1 X 10(-4) M ouabain, whereas ouabain alone produced 34% inhibition. 5. Lysine absorption showed a 71% decrease with 10 micrograms/ml trypsin in a sodium-free medium, and 70% inhibition with Na-free medium alone. 6. The inhibition of lysine absorption after trypsin treatment could be due to inhibition of the active component of lysine transport.     

Probucol as a potent inhibitor of oxygen radical-induced lipid peroxidation and DNA damage: in vitro studies

Probucol, a clinically used cholesterol lowering and antioxidant drug, was investigated for possible protection against lipid peroxidation and DNA damage induced by iron nitrilotriacetate (Fe-NTA) plus hydrogen peroxide (H2O2). Fe-NTA is a potent nephrotoxic agent and induces acute and subacute renal proximal tubular necrosis by catalyzing the decomposition of H2O2-derived production of hydroxyl radicals, which are known to cause lipid peroxidation and DNA damage. Fe-NTA is associated with a high incidence of renal adenocarcinoma in rodents. Lipid peroxidation and DNA damage are the principal manifestation of Fe-NTA induced toxicity, which could be mitigated by probucol. Incubation of renal microsomal membrane and/or calf thymus DNA with H2O2 (40 mM) in the presence of Fe-NTA (0.1 mM) induces renal microsomal lipid peroxidation and DNA damage to about 2.4-fold and 5.9-fold, respectively, as compared to control (P < 0.05). Induction of renal microsomal lipid peroxidation and DNA damage was inhibited by probucol in a concentration-dependent manner. In lipid peroxidation protection studies, probucol treatment showed a concentration-dependent inhibition (10-34% inhibition; P < 0.05) of Fe-NTA plus H2O2-induced lipid peroxidation as measured by thiobarbituric acid reacting species' (TBARS) formation in renal microsomes. Similarly, in DNA damage protection studies, probucol treatment also showed a concentration-dependent strong inhibition (36-71% inhibition; P < 0.05) of DNA damage. From these studies, it was concluded that probucol inhibits peroxidation of microsomal membrane lipids and DNA damage induced by Fe-NTA plus H2O2. However, because the lipid peroxidation and DNA damage studied here are regarded as early markers of carcinogenesis, we suggest that probucol may be developed as a cancer chemopreventive agent against renal carcinogenesis and other adverse effects of Fe-NTA exposure in experimental animals, in addition to being a cholesterol-lowering drug, useful for the control of hypercholestrolemia.     

Properties of the superoxide-generating oxidase of B-lymphocyte cell lines. Determination of Michaelis parameters.

The capacity of three B-lymphocyte cell lines to generate superoxide (O2.-) was examined. The Burkitt lymphoma lines P.3HR-1 and Jijoye gave no response to phorbol 12-myristate 13-acetate (PMA) at 100 ng/ml but produced up to 0.35 nmol of O2.-/min per mg of protein when stimulated with 5 micrograms of PMA/ml; the cell line RPMI 1788 produced Nitro Blue Tetrazolium-positive responses to low PMA concentrations and approx. 0.4 nmol of O2.-/min per mg of protein at 5 micrograms of PMA/ml. Each cell line contained approx. 10 pmol of low-potential cytochrome b (cytochrome b-245)/mg of protein. Homogenates of PMA-activated cells gave 10-20-fold greater rates of O2.- produced per mg of protein. The Km for NADPH varied between approx. 250 microM for P3.HR-1 and RPMI 1788 cell lines and 30.5 +/- 6.5 microM for the Jijoye cell line; the Km values for NADH were higher. Determination of intracellular NADPH concentration showed that this might limit the rate of O2.- production since in each cell line it was at or below the Km concentration.     

Vasoactive intestinal peptide, a singlet oxygen quencher

The neuropeptide vasoactive intestinal peptide (VIP), a highly basic 28-amino acid peptide, has a widespread distribution in the body. The functional specificity of this peptide not only includes its potent vasodilatory activity, but also its role in protecting lungs against acute injury, in preventing T-lymphocyte proliferation and in modulating immune function. We have investigated the possible antioxidant properties of VIP and found that VIP does not have significant O2-, OH., or H2O2 scavenging ability. However, VIP was found to inhibit, in a dose-dependent manner, the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation. 1O2 was produced in photosensitizing systems using rose bengal or methylene blue as sensitizers and was detected as TEMP-1O2 product (TEMPO) by electron paramagnetic resonance (EPR) spectroscopic techniques. The formation of TEMPO signal was strongly inhibited by known singlet quenchers, e.g. beta-carotene, histidine as well as azide, but not by catalase (20 micrograms/ml) which removes H2O2 and mannitol (6 mM) or ethanol (5.9 mM) which remove OH.. Superoxide dismutase (2.5 micrograms/ml) inhibited the photoreaction up to 20% by removing O2- and most probably by blocking the secondary charge transfer pathway of 1O2 formation. These results suggest that the formation of nitroxide radical by 1O2 attack on TEMP may be used as a simple and specific assay for 1O2, and VIP can serve as an effective 1O2 scavenger/quencher, thus it may modulate the oxidative tissue injury caused by this reactive species of oxygen.     

Ferric cacodylate efficiently stimulates growth of rat renal glomerular epithelial cells in vitro

Rat renal glomerular epithelial cells (SGE1 cell line) can be maintained and grown continuously in serum-free medium supplemented with insulin, iron-saturated transferrin (Tr), selenium, bovine serum albumin (BSA), linoleic acid, and epidermal growth factor (EGF). Of the growth supplements used, Tr is essential for proliferation of the cells. In the present study, we describe the use of a unique iron-chelate complex, ferric cacodylate (Fe-Cac), positively charged molecules in neutral buffer, that could almost replace Tr in serum-free culture. It even stimulated the growth of SGE1 cells more efficiently than ferric chloride (FeCl₃) and other iron-chelate complexes, such as ferric nitrilotriacetate (Fe-NTA) and ferric citrate (Fe-Cit). The growth-stimulatory activity of Fe-Cac was exerted at iron concentrations of more than 0.01 g/ml, whereas a 10-fold excess of iron concentration was required with FeCl₃, Fe-NTA and Fe-Cit. We observed that SGE1 cells grew until confluent, then formed hemicysts (domes) in serum-free medium containing Fe-Cac, suggesting that Fe-Cac did not merely permit cell growth but also supported polarization and organization of the cells into a functional epithelial architecture. Moreover, since the stimulatory activity of Fe-Cac was completely abolished by desferrioxamine, a strong iron chelator, it is suggested that iron is crucial for growth of SGE1 cells. When the cells were treated with suramin, an inhibitor of cellular pinocytosis and endocytosis of a large spectrum of ligands including receptor-bound growth factors, growth-stimulatory activity of Tr was inhibited, whereas the activity of Fe-Cac was not affected. These results, taken together, strongly suggest that the growth-stimulatory activity of Fe-Cac is associated with iron delivery into the cells through the cell membrane by diffusion, which is different from Tr receptor-mediated endocytosis. The use of Fe-Cac for investigating iron-regulated cell proliferation is suggested.     

Genotoxicity of 1-nitropyrene and 2,4,7-trinitro-9-fluorenone to mammalian cells

Nitroarenes are ubiquitous environmental pollutants displaying potent genotoxicity in bacterial and mammalian cells. In this study, 2,4,7-trinitro-9-fluorenone (TNF) was more potent than 1-nitropyrene (1-NP) in eliciting genotoxic responses in 4 mammalian cell lines. All 4 cell types were capable of activating the nitroarenes, since no special incubation conditions were required. Inhibition of normal DNA synthesis and cytotoxicity were significantly increased with TNF in a dose range of 0.2-5 micrograms/ml for human teratocarcinoma (PA1) cells, mouse Sertoli (TM4) cells, rat hepatoma (RL12) cells, and human-Chinese hamster ovary (CHO-K1) cells. For 1-NP, a dose range of 10-20 micrograms/ml was required to achieve comparable results for the respective cell lines. Only the RL12 and CHO-K1 cells showed recovery of normal DNA synthesis when TNF or 1-NP was removed from the medium. The other cell types showed little or no recovery up to 42 h after removal of the nitroarene. In exclusively studying TNF, the induction of sister-chromatid exchanges (SCEs) and a delay in cell cycle as monitored by harlequin chromosomes, were observed at a concentration range of 0.003-0.2 microgram/ml in PA1, TM4, and RL12 cells. In CHO-K1 cells, TNF at 0.001-1 microgram/ml was clearly mutagenic at the hprt locus.     

Comparison of the ability of basement membranes produced by corneal endothelial and mouse-derived Endodermal PF-HR-9 cells to support the proliferation and differentiation of bovine kidney tubule epithelial cells in vitro

The proliferation and morphological differentiation of bovine kidney collecting-tubule epithelial cells has been examined as a function of substrata and plasma factors. Collecting kidney tubule explant maintained in vitro gave rise to two distinct cell populations; one was composed mostly of fibroblastic cells whereas the other was epithelioid (EP cells). The proliferation of fibroblastic cells when exposed to serum-supplemented medium was best expressed when cells were maintained on a basement membrane produced by bovine corneal endothelial cells. This basement membrane has a composition, which in previous studies has been shown to favor the proliferation of mesenchymal cells. In contrast, the proliferation of EP cells was best expressed when cells were maintained on a basement membrane produced by the mouse-derived endodermal cell line PF-HR-9 (HR-9-BM). This basement membrane has a biochemical composition very similar to the basement membrane underlying the kidney tubules. Although the fibroblast confluent monolayer maintained on bovine corneal endothelial cell extracellular matrix did not undergo morphogenesis, the confluent monolayer of EP cells maintained on HR-9-BM shows hemicyst formation, suggesting that they were capable of vectorial fluid transport. They also built a complex three-dimensional kidney tubulelike network. Some tubules became grossly visible and floated into the tissue culture medium, remaining tethered to the cell monolayer at either end of the tubule. On an ultrastructural level, the tubules consisted of cells held together with junctional complexes arranged so as to form a lumen. The smallest lumen were bordered by 2-3 cells, and the largest ones by 8-15 cells. The lumens of the larger tubules did contain granular fibrillar and amorphous debris. Low-density EP cell cultures maintained on HR-9-BM could be induced to proliferate at a rate approaching that of cultures exposed to serum when they were exposed to medium supplemented with high-density lipoprotein (HDL, 750 micrograms protein/ml) and transferrin (50 micrograms/ml). When exposed to HDL concentrations equal or lower than 250 micrograms protein/ml, low-density cultures proliferated at a slow rate and readily formed tubulelike structures. This observation indicates that EP cells do not need to reach confluence to undergo morphogenesis, and that HDL, which in the presence of transferrin supports the cell proliferation, can favor their differentiation into tubulelike structures once its concentration becomes limiting for mitogenesis.     

26-hydroxycholesterol-stimulated DNA synthesis in smooth muscle cells and induction of endothelial injury using a coculture technique

We investigated the effects of 0.5, 2.5, and 10 micrograms/ml of cholesterol or 26-hydroxycholesterol on bovine aortic ECs and SMCs. Suppression of viable cell density and cytotoxic changes in both cells were induced by 2.5 and 10 micrograms/ml 26-hydroxycholesterol. ECs were more severely damaged than SMCs in the presence of 26-hydroxycholesterol. Levels of up to 10 micrograms/ml cholesterol had no effect on ECs or SMCs growth or cytotoxicity. Confluent ECs exposed to 2.5 or 10 micrograms/ml of 26-hydroxycholesterol secreted significant amounts of 6-ketoprostaglandin F1 alpha after a 24-hr incubation. An equivalent concentration of cholesterol had no such effect. SMCs cocultured with ECs exposed to 10 micrograms/ml 26-hydroxycholesterol, when compared with an equivalent level of cholesterol, synthesized DNA in significantly greater amounts during a 24-hr incubation. The cocultured ECs incubated in the presence of 2.5 and 10 micrograms/ml 26-hydroxycholesterol were partially detached due to cell death. However, no difference was observed in the DNA content of SMCs cultured without ECs in the presence of cholesterol or 26-hydroxycholesterol. The results suggest that 26-hydroxycholesterol produced not only cytotoxicity to ECs and SMCs but also stimulated DNA synthesis in SMCs through endothelial injury.     

Protective effect of lycopene on lipid peroxidation and oxidative DNA damage in cell culture

A high incidence of cancer has been correlated with chronic iron overload, and carotenoids are of interest as possible anticarcinogens. We have investigated the effect of lycopene on lipid peroxidation and on the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in CV1-P monkey cells exposed to ferric nitrilotriacetate (Fe-NTA) plus ascorbate. Cells supplemented with lycopene (20 pmol/10(6) cells) showed a reduction of 86% in Fe-NTA/ascorbate-induced lipid peroxidation (TBARS). Levels of 8-oxodGuo rose from 1.59+/-0.09 residues/10(6) dGuo in the control cells to 14.02+/-0.41 residues/10(6) dGuo after incubation with (1:4 mM) Fe-NTA/ascorbate (40 microM). Lycopene supplementation decreased in 77% the 8-oxodGuo levels in Fe-NTA/ascorbate-treated cells. These results indicate that lycopene can protect mammalian cells against membrane and DNA damage and possibly play a protective role against tumor promotion associated with oxidative damage.     

Neural tube defects caused by local anesthetics in early chick embryos

The effects of local anesthetics (ketamine HCl, lidocaine HCl, procaine HCl, and tetracaine HCl) on stage 8 (four-somite) chick embryos were investigated. In general, embryos responded to drug treatment in a dose-related manner during the first 6 hr of incubation. Concentrations of 500 micrograms/ml (ca. 2 mM) or higher were embryolethal, whereas 100-200 micrograms/ml (0.1-0.8 mM) preferentially inhibited elevation of neural folds. The latter effect was detectable within 3 hr of treatment and was readily reversible. Tetracaine was the most potent among the four local anesthetics tested at any given dose. Compared to controls, cells in the defective neuroepithelium were less elongated and exhibited smoother apical (luminal) surfaces, thinner microfilament bundles, and less intense actin-specific fluorescence. Furthermore, the effects of local anesthetics (100-200 micrograms/ml) on stage 8 chick embryos were not identical to those of cytochalasin D (0.05 micrograms/ml), colchicine (1 microgram/ml), or ionophore A23187 (25 micrograms/ml), although all treatments produced neural tube defects. Overall results suggest that local anesthetics inhibit closure of the neural tube through their disruptive action on the organization and function of microfilaments in developing neuroepithelial cells.     

Oxygen radicals generated during anoxia followed by reoxygenation reduce the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in human endothelial cell culture

The effect of anoxia and reoxygenation on the synthesis and secretion of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) was studied in primary cultures of human umbilical vein endothelial cells. Sublethal anoxia, determined by trypan blue dye exclusion and lactate dehydrogenase release, was produced by cell culture under a 95% N2, 5% CO2 atmosphere for 2-24 h and was followed by reoxygenation with 95% air, 5% CO2 for 24 or 48 h. Anoxia did not alter the levels of mRNA for t-PA or PAI-1 in the cells or the secretion of t-PA or PAI-1 into the medium. At 24 h, t-PA secreted into conditioned medium was 7.0 +/- 1.4 ng/2 x 10(6) cells (n = 9) and PAI-1 was 300 +/- 13 IU/2 x 10(6) cells (n = 9), whereas the content of t-PA mRNA was 2.2 pg/micrograms of RNA and PAI-1 mRNA was 180 pg/micrograms of RNA. During reoxygenation, however, t-PA antigen and PAI-1 activity as well as mRNA for PAI-1 decreased proportionally to the duration of anoxia, to reach 27 +/- 1.0, 49 +/- 2.0, and 47 +/- 14% of control values, respectively, within 24 h of anoxia. t-PA mRNA also decreased significantly during reoxygenation following anoxia, but the extent could not be accurately quantitated. Addition, during anoxia, of a 200 micrograms/ml concentration of the superoxide anion radical scavenger superoxide dismutase or of a 5 mM concentration of the iron chelator deferoxamine mesylate prevented the subsequent decrease of t-PA antigen during reoxygenation; addition of these compounds during reoxygenation had no effect. Superoxide dismutase, but not deferoxamine mesylate, when added during anoxia prevented the subsequent decrease in PAI-1 activity. These studies suggest that the marked alteration of endothelial cell fibrinolysis during anoxia followed by reoxygenation is most likely mediated by a mechanism dependent on oxygen radicals. Impaired endothelial cell fibrinolysis may contribute to the pathophysiology of ischemia/reperfusion injury.     

Mechanism of cyclosporin A-induced inhibition of prostacyclin synthesis by macrophages

In vitro studies of PG production over a 24 h period by adherent rat peritoneal macrophages activated by serum-opsonized zymosan revealed that CSA (0.3-10 micrograms/ml) caused a dose-related inhibition of PGI2 (assayed as 6-oxo-PGF1 alpha) formation. Indomethacin (IND, 0.01-10 micrograms/ml) and dexamethasone (DEX, 0.01-10 micrograms/ml) also inhibited the PG production in a dose-related manner. When arachidonic acid (10 micrograms/ml) was added together with the inhibitors, there was no change in the level of PGI2 produced by IND-treated cells whilst the PGI2 levels of DEX- and CSA-treated cells were elevated to the control level. Therefore CSA like DEX does not inhibit cyclo-oxygenase activity. However unlike DEX, CSA (1-30 micrograms/ml) caused inhibition of phospholipase A2 (PLA2) activity when assayed on the hydrolysis of a synthetic substrate by pancreatic PLA2 in a cell-free system. The direct inhibition of PLA2 might well be a manifestation of the fundamental activity of CSA on immunocompetent cells.     

Direct protective action of epidermal growth factor on isolated gastric mucosal surface epithelial cells.

Epidermal growth factor (EGF) protects gastric mucosa against acute injury produced by a variety of damaging agents, but the mechanism of its protective action is not clear. Since the surface epithelial cells (SEC) are important component of gastric mucosal defence, we studied whether EGF may directly protect isolated gastric SEC against ethanol injury in vitro, in condition independent of systemic factors and whether endogenous prostaglandins may play a role in EGF's protective action. The isolated SEC from rat gastric mucosa were preincubated in medium only, or medium containing 0.0001-10.0 micrograms/ml of h-rEGF for 15 minutes, and incubated with 8% ethanol for 1 hour. In another study the above experiment was repeated but cells were pretreated with 10(-4) or 10(-5) M indomethacin before EGF treatment. The cell viability was assessed by fast green exclusion test. Incubation of SEC with 8% ethanol significantly reduced SEC cell viability to 50 +/- 2%: EGF 0.1 or 1.0 microgram/ml significantly reduced ethanol induced damage (cell viability 59 +/- 3 and 62 +/- 3% respectively). Pretreatment with 10(-4) M indomethacin (the dose which does not affect SEC viability but inhibit PGE2 and PGI2 generation), significantly reduced protective action of EGF against 8% ethanol injury. EGF 1.0 and 10.0 micrograms/ml alone without ethanol increased PGE2 and 6 keto PGF1 alpha generation by SEC. These studies demonstrated: 1) EGF is able to protect gastric surface epithelial cells directly without mediation by systemic factors. 2) EGF induced protection of SEC may in part be mediated by prostaglandins.     

Effects of vasomotor- and mediator-induced hypotension on bronchomotor tone in swine

We studied the sympathetic neural response on airways to hypotensive stimuli in 19 swine in vivo. The effects of pharmacologically induced hypotension with nitroprusside (NTP) and hypotension elicited by intravenous compound 48/80 (48/80), a mast cell degranulating agent, were compared after equivalent reductions in mean arterial blood pressure (MAP). Reduction of the MAP to 60% of base line with NTP in six swine caused an increase in plasma epinephrine (E) from 60 +/- 28 to 705 +/- 276 pg/ml (P = 0.032) and plasma norepinephrine (NE) from 270 +/- 46 to 796 +/- 131 pg/ml (P = 0.032). Comparable reduction in MAP elicited with 48/80 in six other swine caused a substantially greater increase in both plasma E (9,581 +/- 4,147 pg/ml; P = 0.012 vs. NTP group) and plasma NE (2,239 +/- 637 pg/ml; P = 0.041 vs. NTP group). Catecholamine secretion attenuated mediator-induced changes in lung resistance (RL). In animals receiving 48/80, RL increased from 2.97 +/- 0.31 to 7.44 +/- 0.56 cmH2O.l-1.s. In animals having ganglionic blockade with 7.5 mg/kg iv hexamethonium and beta-adrenergic blockade with propranolol (4.0 mg/kg iv followed by 40 micrograms/kg-1.min-1), comparable doses of 48/80 caused an increase in RL to 18.6 +/- 4.55 cmH2O.l-1.s (P less than 0.04 vs. swine receiving neither hexamethonium nor propranolol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of interleukin 2-dependent cytotoxic T-cell clones. IV. Production of alpha, beta and gamma interferons and interleukin 2 by Lyt-2+ T cells

The production of alpha, beta and gamma interferons (IFN) and interleukin 2 (IL-2) by Lyt-2+-dependent cytotoxic T-cell lines/clones was investigated. Cloned and uncloned T-cell lines specific for H-2Dd or the unique RL male 1 leukemia antigen were studied. After infection with Sendai virus (SV) or Newcastle disease virus (NDV) all cell lines produced IFN-alpha and -beta. Induction of IFN-gamma was attempted with the mitogens Con A, PHA, PWM, SEA, and SEB, with poly(I:C), with antibodies Lyt-1.2, -2.2, and Thy-1.2, or with the target cells Meth A (H-2Dd+) and RL male 1. All mitogens were effective inducers. However, the antibodies and poly(I:C) were not. One uncloned RL male 1-specific cell line CTLL-RP, produced IFN-gamma after induction with RL male 1. Production of IFN-alpha, beta depended on IL-2, whereas production of IFN-gamma did not, although addition of highly purified IL-2 increased IFN-gamma production even in the absence of other inducers. Crude IL-2 inhibited the production of IFN-gamma but not IFN-alpha, beta. In response to mitogens, some T-cell clones also produced IL-2. The results demonstrate that Lyt-2+ cells can produce a broad spectrum of lymphokine activities after appropriate stimulation. Their availability now affords us the opportunity to study the regulation of lymphokine production at the clonal level.     

Normal human bronchial epithelial cells do not show an adaptive response after treatment with N-methyl-N'-nitro-N-nitrosoguanidine

Normal human bronchial epithelial cells cultured in serum-free medium were exposed to low doses o N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to examine whether increased cellular resistance and increased activity of the DNA-repair enzyme O6-methylguanine-DNA methyltransferase could be induced. After treatment with single doses of MNNG a dose-dependent decrease in O6-methylguanine-DNA methyltransferase activity was observed, as expected for this unique repair system. The activity recovered to the starting level in about 24 h when a dose that consumed approximately 65% of the enzyme activity (0.2 micrograms/ml) was given, but did not exceed the activity in the untreated control. Furthermore, treatment every 6 h for 4-5 days with non-toxic concentrations of MNNG (0.04-0.12 micrograms/ml) did not increase O6-methylguanine-DNA methyltransferase activity. Neither was cell survival following a range of challenge doses significantly increased. Our data suggest that human bronchial epithelial cells do not adapt to MNNG.     

Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells.

Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of [3H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of [3H]hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of [3H]thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone.