On the Physical Basis of Biological Signaling by Interface Pulses.

Currently, biological signaling is envisaged as a combination of activation and movement, triggered by local molecular interactions and molecular diffusion, respectively. However, here, we suggest that other fundamental physical mechanisms might play an at least equally important role. We have recently shown that lipid interfaces permit the excitation and propagation of sound pulses. Here, we demonstrate that these reversible perturbations can control the activity of membrane-embedded enzymes without a requirement for molecular transport. They can thus facilitate rapid communication between distant biological entities at the speed of sound, which is here on the order of 1 m/s within the membrane. The mechanism described provides a new physical framework for biological signaling that is fundamentally different from the molecular approach that currently dominates the textbooks.

[Doppler sonography and colour Doppler sonography in the preoperative assessment of anterolateral thigh flap perforators]. / Doppler-Sonografie und Farbdopplersonografie zur präoperativen Perforatordarstellung beim anterolateralen Oberschenkellappen.

BACKGROUND: In the past years the anterolateral thigh flap has become a workhorse flap in the reconstruction of the head and neck region and the extremities. Due to its variable vascular anatomy the preoperative assessment of perforators has been performed using several devices. PATIENTS AND METHODS: In our study we investigated the reliability of Doppler sonography and colour Doppler sonography for this purpose in 14 patients undergoing reconstruction with anterolateral thigh flaps. Colour Doppler sonography identified 31 perforators preoperatively, which were all identified during flap harvest. Doppler sonography found 25 true positive signals, 11 false positive signals and 6 false negative signals. CONCLUSIONS: Both procedures provide a simple and rapid method for the localisation of ALT perforators. However, colour Doppler sonography showed a significantly higher reliability in identifying perforators of anterolateral thigh flaps, thus enabling flap design around preoperatively confirmed perforators.

Decreased zinc toxicity resulting from doxorubicin without increased GSSG export in three human lung cell lines.

Zinc-mediated cytotoxicity is recognized, at least in part, by a decrease of reduced glutathione (GSH) and an increase in the oxidized form of glutathione (GSSG). Doxorubicin is a common inducer of multidrug-resistance-associated proteins and such proteins might, furthermore, be associated by an increased GSSG export rate. Therefore, zinc-mediated toxicity should be abolished after doxorubicin pretreatment. In the present study, zinc toxicity was characterized by methionine incorporation, glutathione content, and the GSSG/GSH ratio. Experiments were performed in three established lung cell lines comparing doxorubicin-pretreated cells with controls. Zinc-mediated toxicity was significantly decreased after pretreatment with doxorubicin as assessed by methionine-incorporation inhibition, GSH depletion, and/or GSSG increase in the two nonmalignant cell lines. Unexpectedly, zinc-associated GSSG export was not increased after doxorubicin pretreatment. This inconsistency might be explained as a result of a decreased zinc content in these cells, probably because of an increased export rate of zinc. The findings are in contradiction to the opinion of metal excretion by multidrug-resistance-associated proteins, matched to GSH conjugate excretion, as it is discussed for cadmium, for example.

Decreased GSSG reductase activity enhances cellular zinc toxicity in three human lung cell lines.

Cellular reduced glutathione (GSH) levels have been identified as an essential determinant in zinc-induced cytotoxicity. However, cytotoxic effects of zinc have also been observed without depletion of GSH stores. In a previous study, the intracellular activity of GSSG reductase (GR) has come into focus (Walther et al. 2000, Biol Trace Elem Res 78:163-177). In the present paper we have tried to address this issue more deeply by inhibiting the activity of cellular GR without any appreciable decreases of cellular glutathione. In three pulmonary cell lines, GR activity was inhibited in a dose-dependent manner by the alkylating agent carmustine (BCNU), a known inhibitor of GR. Cells were pretreated with BCNU for 14 h, followed by exposure to various concentrations of zinc chloride. Then we determined the incorporation of radiolabelled methionine (to assess protein synthesis), and measured the GSH and oxidized glutathione (GSSG) levels. Additionally, GR activity of controls was measured. IC(50) values for zinc-induced inhibition of methionine incorporation, as well as GSH contents, was strongly correlated to the decreased GR activity. These results firmly suggest that GR is an important factor in the event chain of zinc cytotoxicity. Together with the results from our previously cited study where impaired regeneration of GSH levels were accompanied by a decrease in total cellular glutathione (GSH + GSSG) we conclude that GSSG itself is an important effector in zinc cytotoxicity.

Effect of low level zinc pretreatment on zinc-mediated toxicity in different lung cell lines.

Reduced toxicity of high zinc exposure was observed after pretreatment of various lung cells with nonlethal zinc concentrations. This effect became significant when various parameters of cytotoxicity were assessed (e.g., inhibition of protein synthesis, depletion of reduced glutathione [GSH], increase of oxidized glutathione [GSSG], release of lactate dehydrogenase [LDH]). Similar protective effects by zinc have already been shown by several investigators for a variety of toxicity studies dealing with cadmium, in vitro and in vivo. Zinc-induced toxicity has been linked to glutathione metabolism and cellular GSH contents. Activity of glutathione reductase (GR) and rates of glutathione synthesis were identified as determinants of zinc (cyto)toxicity. However, these variables were virtually unaffected in our adapted cells. Consequently, another variable appears to be crucial for modulating cellular suscepticibility in zinc pretreated cells. Protection in our cells was achieved by pretreatment with 80-120 micromol/L zinc chloride for 24-72 h, roughly 10-fold more zinc in the medium than is normally found in human plasma. Protection was not observed when the cells were concomitantly exposed to cycloheximide, an inhibitor of protein synthesis, or actinomycin D, an inhibitor of RNA synthesis, but it was found in the presence of amanitin, an inhibitor of mRNA synthesis. It is therefore concluded that the altered zinc tolerance of pretreated cells is not attributable to the induction of metallothionein.

Effects of zinc chloride on glutathione and glutathione synthesis rates in various lung cell lines.

Zinc-mediated toxicity has been linked to cellular glutathione (GSH) contents. In this study, effects of zinc on cellular GSH content, glutathione reductase (GR) activity, and GSH synthesis were investigated. In all cell lines tested, decreases in cellular GSH content and GR activity as well as an increase in oxidized glutathione (GSSG) were found after incubation of cells with zinc chloride. These effects were dose- and time-dependent. Changes in GR activities were earliest affected and were most marked compared with the other parameters examined. Decrease of enzyme activity was not due to a decrease in the cosubstrate NADPH. In A549 and L2 cells, initial increases in GSH synthesis rates occurred up to about 175% of control. Later, GSH synthesis decreased to levels below controls. In 16Lu cells, GSH synthesis decreased after 2 h of zinc exposure. No transient increase was found in this cell line. Measurement of ATP content did not show any influence of zinc on cellular ATP. Lactate dehyrogenase leakage, a marker of a clear cytotoxic effect, occurred after 6 h of zinc treatment in the non-malignant cells examined, and after 16 h in malignant A549 cells. We assume the inhibition of GR activity and the associated increase of GSSG could possibly represent a main zinc-mediated toxic cellular effect.

Lack of effects of hydrocortisone pretreatment on zinc-induced changes in protein assemble.

Inhalational zinc intoxication may lead to the development of acute respiratory distress syndrome (ARDS). Pharmacological treatment of ARDS is based on glucocorticoids, while the efficiency of glucocorticoid treatment is discussed controversially. Glucocorticoid pretreatment of lung cell lines is known to cause disparate effects with regard to zinc susceptibility. Both substances are known to each interact with protein metabolism. In the present study, zinc effects were examined on hydrocortisone (HC)-pretreated lung cell lines by detection of content and synthesis of different proteins after two-dimensional (2D) gel electrophoresis. (1) In HC- pretreated fibroblast-like 11Lu and alveolar epithelial L2 cells, no zinc-mediated changes after silver staining of 2D gels were seen. Few differences occurred in HC-pretreated alveolar epithelial A549 cells that might be explained by the appearance of heat shock proteins (hsp) after zinc exposure. (2) In autoradiographs after 35S-Met incorporation only in 11Lu cells, small differences occurred after HC treatment as compared to controls without HC. (3) All cell lines tested demonstrated the same zinc-mediated changes in autoradiographs with a nearly complete loss of synthesized proteins and an appearance of a few new spots. These changes were reversible in all cell lines after washing out of external zinc. The new spots were transiently expressed for a few hours after zinc exposure. (4) The overall effect of HC pretreatment was rather unimpressive. The virtual lack of major effects does not support the hypothesis that a gross interaction between glucocorticoids and zinc at the cellular protein synthesis level would be an important mechanism of influence in zinc-induced lung injury.

Zinc toxicity in various lung cell lines is mediated by glutathione and GSSG reductase activity.

In a previous work, it was shown that in cells after a decrease of cellular glutathione content, toxic zinc effects, such as protein synthesis inhibition or GSSG (glutathione, oxidized form) increases, were enhanced. In this study, zinc toxicity was determined by detection of methionine incorporation as a parameter of protein synthesis and GSSG increase in various lung cell lines (A549, L2, 11Lu, 16Lu), dependent on enhanced GSSG reductase activities and changed glutathione contents. After pretreatment of cells with DL-buthionine-[R,S]-sulfoximine (BSO) for 72 h, cellular glutathione contents were decreased to 15-40% and GSSG reductase activity was increased to 120-135% in a concentration-dependent manner. In BSO pretreated cells, the IC50 values of zinc for methionine incorporation inhibition were unchanged as compared to cells not pretreated. The GSSG increase in BSO pretreated cells by zinc was enhanced in L2, 11Lu, and 16Lu cells, whereas in A549 cells, the GSSG increase by zinc was enhanced only after pretreatment with the highest BSO concentration. Inhibition of GSSG reductase in alveolar epithelial cells was observed at lower zinc concentrations than needed for methionine incorporation inhibition, whereas in fibroblastlike cells, inhibition of GSSG reductase occurred at markedly higher zinc concentrations as compared to methionine incorporation inhibition. These results demonstrate that GSSG reductase is an important factor in cellular zinc susceptibility. We conclude that reduction of GSSG is reduced in zinc-exposed cells. Therefore, protection of GSH oxidation by various antioxidants as well as enhancement of GSH content are expected to be mechanisms of diminishing toxic cellular effects after exposure to zinc.

Influence of glutathione on zinc-mediated cellular toxicity.

The effect of zinc on various pulmonary cell lines has been studied by measuring the depletion of total cellular glutathione after exposure to zinc(II) chloride at different concentrations. Total cellular glutathione (cGS) was measured at 31+/-3 nmol/mg, 3.8+/-0.6 nmol/mg, and 3.7+/-1.2 nmol/mg protein in A549, L2, and 11Lu cells, respectively. After treatment with buthionine sulfoximine (BSO), the cGS levels decreased by 20% in A549 cells and below 0.2 nmol/mg in L2 and 11Lu cells. Exposure of A549 cells to 25-200 microM ZnCl2 for 4 h alone decreased the cGS content to 60-80%. There was little additional effect in BSO-pretreated cells. In L2 and 11Lu cells, the decrease of cGS was 70-85% following exposure to 15-150 microM ZnCl2 for 2 h. If BSO was also used, the decrease in cGS was 85-95% in L2 cells and 75-85% in 11Lu cells. Exposure to 25-250 microM ZnCl2 for 2 h diminished protein synthesis as determined by radiolabeled methionine incorporation, with half-maximum inhibition (EC50) from 40-160 microM ZnCl2. To attain similar EC50 values in BSO-pretreated cells, only about half the zinc concentrations were required as compared to cells without pretreatment. The decrease of cGS was accompanied by an increased ratio of oxidized: reduced glutathione that was more pronounced in cells with low glutathione content.

A novel apparatus for the exposure of cultured cells to volatile agents.

This article presents a novel exposure apparatus that allows the exposure of cultured cells to volatile chemicals, e.g., inhalation anesthetics. The apparatus consists of an exposure chamber and a tightly linked vaporizer unit with pumps and valves allowing adjustable fluxes of mixtures of test chemicals and carrier gas under open and closed-circuit conditions. The exposure chamber uses commercially available cell culture flasks and accommodates up to 12 flasks simultaneously. Both modules fit into a standard culture incubator. The exposure chamber may be mounted onto an oscillating axis to tilt the cultures periodically forth and back, thus allowing direct contact of the cells with test atmosphere. The vaporizer unit is connected to a personal computer which lets the experimenter set the "open" and "close" intervals of individual valves thereby controlling the composition and flow rate of the test gas mixture. The vapor concentration of test chemicals can be monitored at the inlet and outlet using infrared photodetectors or mass spectrometers. Computer-aided processing of exposure protocols allows unattended runs. Exposure protocols can be scripted and stored on disk, thus ensuring interexperimental reproducibility of complex exposure profiles. As an application example, the effect of three volatile anesthetics, halothane, enflurane, and isoflurane, on the viability of three commercially available cell lines (A549--human lung carcinoma, HTC-rat hepatoma, MDCK--Madin-Darby canine kidney) was investigated. After exposure to haloalkyl vapors (3%) for 6 and 24 h, respectively, significantly increased LDH levels versus controls, indicating cellular membrane damage, were detected in A549 and hepatoma cells after exposure for 24 h. Hepatoma cells showed a significant LDH release also after 6 h exposure to isoflurane. On the other hand, LDH release from MDCK cells was not significantly different from controls even after 24 h of continuous exposure to any of the tested anesthetics.

Are we ready to replace dimercaprol (BAL) as an arsenic antidote?

1 Dimercaprol (BAL), 2,3-dimercaptopropanesulphonate sodium (DMPS) and meso-2,3-dimercaptosuccinic acid (DMSA) are effective arsenic antidotes, but the question which one is preferable for optimal therapy of arsenic poisoning is still open to discussion. Major drawbacks of BAL include (a) its low therapeutic index, (b) its tendency to redistribute arsenic to brain and testes, for example, (c) the need for (painful) intramuscular injection and (d) its unpleasant odour. 2 The newer antidotes DMPS and DMSA feature low toxicity and high therapeutic index. They can be given orally or intravenously due to their high water solubility. While these advantages make it likely that DMPS and DMSA will replace BAL for the treatment of chronic arsenic poisoning, acute intoxication-especially with lipophilic organoarsenicals-may pose a problem for the hydrophilic antidotes, because their ionic nature can adversely affect intracellular availability. 3 This article focuses on aspects dealing with the power of BAL, DMPS, and DMSA to mobilize tissue-bound arsenic in various experimental models, such as monolayers of MDCK (= Madin-Darby canine kidney) cells from dog kidney, isolated perfused liver from guinea-pigs, and perfused jejunal segments from rat small intestine. 4 The results show that hydrophilic DMPS and DMSA may fail to rapidly and completely remove arsenic that has escaped from the extracellular space across tight epithelial barriers. However, owing to their low toxicity, which allows larger doses to be applied, and the potential modification of their pharmacokinetics by means of inert oral anion-exchange resins, DMPS and DMSA may advantageously replace BAL whenever intervention time is not critical. With severe intoxication by organic arsenicals, when the point-of-no-return is a limiting factor, BAL may still have a place as an arsenic antidote.

Reversal of oxophenylarsine-induced inhibition of glucose uptake in MDCK cells.

It has been shown that oxophenylarsine (PhAsO) inhibits glucose uptake in MDCK cells. In addition to the known impairment of cellular energy metabolism, this inhibition may contribute to the acute toxicity of trivalent organic arsenicals. We have investigated the effect of BAL, DMPS, DMSA, and other sulfur compounds on cellular incorporation of [U-14C]PhAsO and their efficacy to revert PhAsO-induced inhibition of glucose uptake. In the presence of [U-14C]PhAsO (2 microM), the radiolabel was steadily accumulated by the cells over 150 min without any signs of severe cell damage (e.g., altered morphology, increased LDH release). A notable decrease of cellular ATP was only observed at 150 min, whereas within 30 min uptake of D-[6-(14)C]glucose was reduced to 40% of controls. When BAL, DMPS, or DMSA was added after 30 min, the inhibition of glucose uptake was reversed, accompanied by a decrease in cell-associated radiolabel from [U-14C]-PhAsO. Water-soluble DMPS and DMSA required longer times than BAL for comparable effects. 2,3-Bis(acetylthio)propanesulfonamide, a thioester derivative, and dithiothreitol, a 1,4-dithiol, were effective only with the highest concentration tested (200 microM). 2-Mercaptoethanol neither reversed inhibition of glucose uptake nor influenced [U-14C]PhAsO incorporation. Our results show that inhibition of glucose uptake is a very early event in PhAsO cytotoxicity which occurs before any decrease of cellular energy metabolism and/or full cellular loading with arsenic comes into effect. The more rapid onset of action of lipophilic BAL compared to PhAsO action.

Influence of 2,3-dimercaptopropanol and other sulfur compounds on oxophenylarsine-mediated inhibition of glucose uptake in MDCK cells.

Trivalent monosubstituted organoarsenicals, e.g., oxophenylarsine (PhAsO), exert various detrimental effects on mammalian cells. In addition to their well known interference with pyruvate and ketoglutaric acid oxidation, the effect on other cellular functions such as uptake of glucose may contribute to their acute toxicity. Different effects of PhAsO on insulin-stimulated and insulin-independent uptake of hexoses in various tissues have been reported. It has been shown previously that PhAsO inhibits the stereospecific uptake of glucose in MDCK cells. In this work, the insulin dependence of glucose uptake in these cells and the effects of 2,3-dimercaptopropanol (BAL), dithiothreitol (DTT) and 2-mercaptoethanol (ME) on PhAsO-induced inhibition of glucose uptake were investigated. A 200 mumol l-1 concentration of insulin had no measurable effect on cellular 14C accumulation from D-[6(-14)C]glucose, indicating an insulin-independent hexose transport system. In the presence of 2 mumol l-(-1) of PhAsO, glucose uptake was lowered to less than 50% of controls within 30 min. Greater inhibition was observed with higher concentrations of PhAsO, but cell viability as assessed by formazan formation started to decrease at concentrations > or = to 5 mumol l-1, especially after longer exposure times. When BAL was added in a ten-fold molar excess 30 min after beginning incubation with PhAsO (2 mumol l-1, virtually complete recovery of inhibited glucose uptake occurred within 10 min after addition. ME at up to a 100-fold molar excess over arsenic had no influence on the inhibition of glucose uptake within 120 min after addition.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of glucose on the toxicity of oxophenylarsine in MDCK cells.

Trivalent arsenicals like oxophenylarsine (PhAsO) inhibit cellular pyruvate dehydrogenase, thus leading to a drop of acetylCoA formation and a slow-down of the citric acid cycle. Glucose may protect cells from arsenic toxicity, because increased glycolysis may prevent fatal shortage of ATP. On the other hand, PhAsO has been shown to inhibit glucose uptake in Madin-Darby canine kidney (MDCK) cells. We have investigated the effect of PhAsO on viability, ATP levels and glucose uptake of MDCK cells in the presence of normal (5 mmol/l) and low (0.01 mmol/l) glucose concentrations. At normal as well as at low glucose concentrations, cell viability as assessed by formazan formation was not affected by PhAsO concentrations up to 2 mumol/l within 3 h of observation. At higher PhAsO concentrations viability was diminished earlier and more pronounced in the presence of low glucose concentrations. 10 mumol/l PhAsO induced a drastic drop of ATP within 30 min which was followed by an almost complete loss of viable cells after 180 min in the presence of low glucose concentrations, while at normal glucose levels no influence on ATP contents or on cell viability was detected within 60 min of incubation. On the other hand, glucose uptake, determined as 14C accumulation by cells incubated for 10 min with D-[6-14C]-glucose, was inhibited by PhAsO at low as well as at normal glucose concentrations in a dose dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of DMPS and various adsorbents on the arsenic excretion in guinea-pigs after injection with As2O3.

The present experiments were performed to test the possibility of interrupting the enterohepatic circulation of arsenic (As). Therefore the efficacy of adsorbents to bind As and/or As-DMPS adducts in vitro and their effect on the excretion of As into the feces and urine in vivo were investigated after injection of As2O3 and DMPS in guinea-pigs. The adsorbents bentonite, activated charcoal or colestyramine, respectively, were tested. Only slight binding of 73As (< 5% of the 73As dose) was observed to all adsorbents in vitro. After addition of DMPS, a good binding was found for 73As to colestyramine (50%) or activated charcoal (60%), respectively. However, the 73As-DMPS adduct was removed from the activated charcoal during washing. In the first in vivo experiment, male guinea-pigs (n = 4/group) received As2O3 [0.02 mmol As(III)/kg s.c. labelled with a tracer dose of 73As(III) (0.14 kBq/g)], 30 min later DMPS (0.1 mmol/kg i.p.) and by gastric tube (10 ml/kg body wt) either saline, bentonite (1 g/kg), activated charcoal (1 g/kg) or colestyramine (0.2 g/kg), respectively. Urine and feces were collected for 24 h. No increase in 73As excretion into the feces was observed after administration of DMPS and all adsorbents, compared to control animals. In the second in vivo experiment male guinea-pigs (n = 4/group) received the same As2O3 (+ 73As)- and DMPS dose. In addition, with a gastric tube (10 ml/kg) saline, colestyramine (0.2 g/kg), DMPS (0.1 mmol/kg), or the combination of DMPS (0.1 mmol/kg) + colestyramine (0.2 g/kg) were administered according to the scheme given in the following table. The amount of feces excreted did not differ between groups. Excretion of 73As within the feces during the first 12 h after As injection is shown in the following table (mean +/- SEM). The same amount of 73As (34% of the 73As dose) was excreted into the urine from animals in groups 4 and 5 during this time. Obviously, the combined oral administration of DMPS + colestyramine markedly enhanced fecal excretion of As mobilized by DMPS i.p. It is suggested that interruption of enterohepatic circulation of As may be a valuable adjunct in the treatment of As poisoning.

Influence of glucocorticoids and activated charcoal on the lethality of rats after acute poisoning with T-2 toxin, diacetoxyscirpenol, or roridin A.

Lethal doses of the trichothecene mycotoxins T-2 toxin (1.5 mg/kg), diacetoxyscirpenol (DAS; 2.3 mg/kg) or roridin A (1.3 mg/kg) were intravenously administered to rats. When rats were treated with either activated charcoal (Superchar liquid, Norit A; 1 g/kg, po) or dexamethasone (8 mg/kg, iv) 30 min after poisoning with one of the trichothecenes, lethality was only marginally reduced. However, when the combination of activated charcoal (Superchar liquid or Norit A) and dexamethasone was administered, the survival rate of animals after 30 days was significantly enhanced by up to 50%. Comparison between 2 preparations of activated charcoal, Norit AR with a surface area of 1,000 m2/g and Superchar liquidR with a surface area of 3,000 m2/g, each in combination with dexamethasone, revealed no difference in their therapeutic efficacy. Prednisolone (60 mg/kg) was as effective as dexamethasone (8 mg/kg), each administered with activated charcoal, in preventing death in acute T-2 toxicosis.

Effect of various antidotes on the biliary and intestinal excretion of arsenic in situ and into the feces in vivo in guinea-pigs after injection of As2O3.

The effect of various antidotes on the excretion of arsenic into the feces in vivo and on the biliary and enteric excretion in situ was investigated on segments of jejunum and colon in anesthetized guinea-pigs using the pendular perfusion technique, according to Henning and Forth (1982). In the in situ experiments guinea-pigs received As2O3 (0.02 mmol As(III)/kg) and 30 min later, British-Anti-Lewisite (BAL), dimercaptopropanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA) or 2,3-bis-(acetylthio)propanesulfonamide (BAPSA) (0.1 or 0.7 mmol/kg each) into the jugular vein. In the in vivo experiments guinea-pigs received As2O3 s.c. (same dose as above) and 30 min later the same antidotes (0.1 mmol/kg i.p.). The feces were collected for 24 h and the arsenic content measured. During the 60-min perfusion period the amount of arsenic excreted into the jejunum or colon was only 3% or 0.4% of the dose administered, respectively. Of the arsenic dose, 8% was found in the bile. None of the antidotes had an effect on the arsenic excretion into the jejunum or colon. No change in biliary excretion was found in animals treated with BAL, 0.1 or 0.7 mmol/kg, respectively. DMSA, BAPSA or DMPS, 0.1 mmol/kg, increased the biliary excretion of arsenic to 14, 33, or 43% of the dose administered and after 0.7 mmol/kg to 29, 37, or 42%, respectively. Furthermore, a significant increase (P > 0.05) was found for the bile/blood concentration ratio in the following order: control < BAL < DMSA < BAPSA approximately DMPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated rat kidney tubules as a screening system for arsenic antidotes.

Gluconeogenesis is one of the metabolic pathways severely affected in acute arsenic poisoning. We have studied gluconeogenesis in isolated kidney tubules of male Sprague-Dawley rats to screen various sulfur compounds for antidotal properties against inorganic and organic arsenicals. Freshly prepared kidney cells from starved rats synthesized glucose from added pyruvate (10 mmol/liter) at a rate of 9.74 +/- 0.90 nmol/min/mg protein (mean +/- SD; n = 61). Gluconeogenesis was inhibited almost 90% in the presence of phenylarsonate (700 mumol/liter), arsenate (350 mumol/liter), arsenite (30 mumol/liter), or PhAsO (1 mumol/liter). mumol/liter). With effective antidotes the rate of gluconeogenesis was restored to almost control values within 10 min. Among 21 sulfur compounds tested, only BAL, DMPS, and DMSA were effective in PhAsO poisoning. With inorganic arsenic also DTE and DTT restored the rate of glucose formation. The observed in vitro efficacies were in good agreement with in vivo results obtained with male NMRI mice severely poisoned with arsenite (As2O3, 20 mg/kg approximately 0.2 mmol As/kg) or PhAsO (3.4 mg/kg approximately 0.02 mmol As/kg). We conclude that isolated kidney tubules are a useful in vitro screening system (a) to compare the metabolic toxicity of various arsenicals and (b) to evaluate potential antidotes.

Influence of inorganic and organic arsenicals on intestinal transfer of nutrients.

The effects of organic (oxophenylarsine, PhAsO 2.5-50 mumol/l) and inorganic arsenicals (As2O3 2.5-250 mumol/l; As2O5 2.5-2500 mumol/l) on intestinal transfer of water, sodium, glucose and leucine was investigated in vitro using isolated jejunal segments of male Sprague Dawley rats. All three arsenicals decreased in a dose-dependent manner the transfer of water, sodium, glucose and leucine. At the highest concentrations investigated the amount absorbed was reduced to some 10-20% of the respective control values. For both glucose and leucine the concentration ratio between absorbate and perfusate was about 3.5-4 in controls. It decreased to about 2 for leucine and to near unity for glucose. As assessed from the concentration ratio between intestinal tissue and perfusate the arsenic compounds inhibited the uptake of glucose and leucine into the tissue. There was a marked difference with respect to the potency of arsenicals, PhAsO being about 10 times more potent than As2O3 which in turn was about 5 times more potent than As2O5.

Influence of organic and inorganic arsenicals on glucose uptake in Madin-Darby canine kidney (MDCK) cells.

The effect of organic (oxophenylarsine; PhAsO) and inorganic (arsenite) arsenicals on the availability of glucose to Madin-Darby canine kidney (MDCK) cells was investigated. The MDCK cells revealed stereospecific D-glucose uptake which was inhibited by both arsenicals in a time- and concentration-dependent manner. After 10 min (37 degrees C), the effects on D-glucose and 2-deoxy-D-glucose accumulation were analogous, suggesting an impaired hexose uptake. With arsenite, 0.5-1 mmol dm-3 were required for half-maximum inhibition (IC50), whereas PhAsO inhibited glucose uptake in the micromolar range (IC50 5-30 mumol dm-3). Under these conditions neither cell morphology nor cellular viability was affected. After 60 min, however, the inhibition of glucose utilization was paralleled by the formation of blebs, detachment of the monolayer and a loss of cellular viability as confirmed by dye exclusion, lactate dehydrogenase and potassium release. It is concluded that inhibition of glucose uptake may contribute to the acute toxicity, especially of organic arsenicals, by further aggravating the depletion of intracellular carbohydrates.