Effects of Arsenic Compounds on Microminerals Content and Antioxidant Enzyme Activities in Rat Liver.

Interactions of arsenic with essential trace elements may result in disturbances on body homeostasis. In the present study, we aimed to investigate the effects of different arsenic compounds on micromineral content and antioxidant enzyme activities in rat liver. Male Wistar rats were randomly divided into five groups and exposed to sodium arsenite and sodium arsenate at 0.01 and 10 mg/L for 8 weeks in drinking water. The concentration of arsenic increased in the liver of all arsenic-exposed animals. The proportion of zinc and copper increased in animals exposed to 0.01 mg/L sodium arsenite. In addition, these animals presented a reduction in magnesium and sodium content. Superoxide dismutase activity decreased mainly in arsenite-exposed animals, whereas catalase activity decreased in animals exposed to 10 mg/L sodium arsenate. Further, exposure to sodium arsenate at 10 mg/L altered copper and magnesium content in the liver, and reduced total protein levels. Overall, both arsenic compounds altered the liver histology, with reduction in the proportion of cytoplasm and hepatocyte, and increased the percentage of sinusoidal capillaries and macrophages. In conclusion, our findings showed that oral exposure to arsenic compounds disturbs the trace elements balance in the liver, especially at low concentration, altering enzymatic and stereological parameters. We concluded that despite the increase in trace elements content, the antioxidant enzyme activities were downregulated and did not prevent morphological alterations in the liver of animals exposed to both arsenic compounds.

Effects of Sodium Arsenite and Arsenate in Testicular Histomorphometry and Antioxidants Enzymes Activities in Rats.

The main source of environmental arsenic exposure in most countries of the world is drinking water in which inorganic forms of arsenic predominate. The present study was aimed to test the impact of two different compounds of inorganic arsenic in histomorphometric and enzymatic parameters in the testes by oral exposition. Adult Wistar male rats were exposed to sodium arsenite and arsenate in drinking water, testing for each chemical form the concentrations of 0.01 and 10 mg/L per 56 days. The animals intoxicated with arsenic, mainly sodium arsenite, showed reduction in the percentage of seminiferous epithelium and in proportion and volume of Leydig cells. Moreover, there was an increase in the percentage of tunica propria, lumen, lymphatic space, blood vessels, and macrophages. The activity of superoxide dismutase (SOD) did not change among the groups. However, the activity of catalase (CAT) decreased in animals exposed to both arsenic compounds. In addition, the higher concentration of arsenic, mainly as sodium arsenite, caused vacuolization in the seminiferous epithelium. The body and testes weight as well as testosterone concentration remained unchanged among the groups. In conclusion, exposition to arsenic, mainly as sodium arsenite, caused alteration in histomorphometric parameters and antioxidant defense system in the testes.

Purification, properties, and kinetic studies of cytoplasmic malate dehydrogenase from Taenia solium cysticerci.

Malate dehydrogenase (L: -malate: NAD oxidoreductase, EC 1.1.1.37) from the cytoplasm of Taenia solium cysticerci (cMDHTs) was purified 48-fold through a four-step procedure involving salt fractionation, ionic exchange, and dye affinity chromatography. cMDHTs had a native M (r) of 64,000, while the corresponding value per subunit, obtained under denaturing conditions, was 32,000. The enzyme is partially positive, with an isoelectric point of 8.7, and had a specific activity of 2,615 U mg(-1) in the reduction of oxaloacetate. The second to the 21st amino acids from cMDHTs N-terminal group were P G P L R V L I T G A A G Q I A Y N L S. This sequence is 100% identical to that of Echinococcus granulosus. Basic kinetic parameters were determined for this enzyme. The optimum pH for enzyme reaction was at 7.6 for oxaloacetate reduction and at 9.6 for malate oxidation. K (m) values for oxaloacetate, malate, NAD, and NADH were 2.4, 215, 50, and 48 microM, respectively. V (max) values for the substrates and cosubstrates as described above were 1,490, 87.8, 104, and 1,714 micromol min(-1) mg(-1). Several NAD analogs, structurally altered in either the pyridine or purine moiety, were observed to function as coenzymes in the reaction catalyzed by the purified malate dehydrogenase. cMDHTs activity was uncompetitive inhibited by arsenate for both the forward (Ki = 8.2 mM) and reverse (Ki = 77 mM) reactions. The mechanism of the cMDHTs reactivity was investigated kinetically by the product inhibition approach. The results of this study are qualitatively consistent with an Ordered Bi Bi reaction mechanism, in which only the coenzymes can react with the free enzyme.

Phosphate increases mitochondrial reactive oxygen species release.

The effects of inorganic phosphate (Pi), the main intracellular membrane permeable anion capable of altering mitochondrial pH gradients (Delta pH), were measured on mitochondrial H2O2 release. As expected, Pi decreased Delta pH and increased the electric membrane potential (Delta Psi). Mitochondrial H2O2 release was stimulated by Pi and also by its structural analogue arsenate. However, acetate, another membrane-permeable anion, did not stimulate mitochondrial H2O2 release. The stimulatory effect promoted by Pi was prevented by CCCP, which decreases transport of Pi across the inner mitochondrial membrane, indicating that Pi must be in the mitochondrial matrix to stimulate H2O2 release. In conclusion, we found that Pi and arsenate stimulate mitochondrial reactive oxygen release, an effect that may contribute towards oxidative stress under conditions such as ischemia/reperfusion, in which high-energy phosphate bonds are hydrolyzed.

Cytotoxic effect of three arsenic compounds in HeLa human tumor and bacterial cells.

Numerous epidemiological studies suggest that arsenic (As) compounds are carcinogens, however, recent data have renewed the interest in their anticarcinogenic properties. The cytotoxic effects of three arsenic compounds were assessed: sodium arsenite, sodium arsenate and sodium cacodylate, representing the trivalent and pentavalent species of arsenic, along with a dimethylated pentavalent arsenic species. HeLa cells and Salmonella typhimurium (strains TA98 and TA100) were exposed to As compounds and the cytotoxic effects were evaluated. Alterations on RNA and DNA synthesis in HeLa cells were also examined. All arsenic compounds produced a dose-dependent inhibition on colony formation and DNA synthesis in HeLa cells, yet any of them significantly influenced RNA synthesis in these cells. No evidence of arsenic-induced mutagenicity or antimutagenicity was observed using the Ames assay. In bacterial cells, only sodium arsenite caused a dose-dependent inhibition of colony formation.Collectively, these results indicate that in both, HeLa and S. typhimurium cell systems, only trivalent sodium arsenite can act as an effective inhibitor of cell growth. The possible mechanism(s) of the cytotoxic effect of arsenite in these two different cell systems might be due to its reactivity with intracellular sulfhydryl groups.

Cytosolic [Ca(2+)] modulates basal GLUT1 activity and plays a permissive role in its activation by metabolic stress and insulin in rat epithelial cells.

The aim of this work was to investigate the role of cytosolic free calcium ([Ca(2+)]c) in the stimulation of GLUT1 by metabolic stress and insulin. Chelation of [Ca(2+)]c with bapta, introduced in rat liver epithelial Clone 9 cells in the acetoxymethyl (AM) form, decreased their basal rate of 2-deoxyglucose uptake in a dose-dependent fashion. Maximal inhibition at 75 microM bapta was by 38 +/- 8% (n = 8). The effect was partially reversed by ionomycin. Basal sugar uptake was also decreased by lowering extracellular [Ca(2+)] in ionomycin-permeabilized cells. Increasing [Ca(2+)]c over its resting level of 168 +/- 32 (n = 27) had no affect on sugar uptake. Chelation of [Ca(2+)]c did not change the abundance of surface GLUT1 and had no significant effect on the affinity of GLUT1 for sugars. In addition, calcium chelation abolished the activation of GLUT1 by azide, arsenate, 2,4-dinitrophenol and insulin. However, [Ca(2+)]c did not increase in the presence of azide. We conclude that [Ca(2+)]c, near or below its resting level, modulates GLUT1 activity over a considerable range and plays a permissive role in the activation of the carrier by metabolic stress and insulin.

Interactions of arsenate, sulfate and phosphate with yeast mitochondria.

In the presence of K(+), addition of ATP or ethanol to yeast mitochondria triggers the depletion of the transmembrane potential (DeltaPsi) and this is prevented by millimolar concentrations of phosphate (PO(4)). Different monovalent and polyvalent anions were tested for their protective effects on mitochondria from Saccharomyces cerevisiae. Only arsenate (AsO(4)) and sulfate (SO(4)) were as efficient as PO(4) to protect mitochondria against the K(+) mediated swelling, depletion of the DeltaPsi, and decrease in the ratio of uncoupled state to state 4 respiration rates. Protection by PO(4), SO(4) or AsO(4) was inhibited by mersalyl, suggesting that these anions interact with a site located in the matrix side. In addition, the effects of SO(4) and AsO(4) on the F(1)F(0)-ATPase were tested: both SO(4) and AsO(4) inhibited the synthesis of ATP following competitive kinetics against PO(4) and non-competitive kinetics against ADP. The mersalyl sensitive uptake of (32)PO(4) was not inhibited by SO(4) or AsO(4), suggesting that the synthesis of ATP was inhibited at the F(1)F(0)-ATPase. The hydrolysis of ATP was not inhibited, only a stimulation was observed when AsO(4) or sulfite (SO(3)) were added. It is suggested that the structure and charge similarities of PO(4), AsO(4) and SO(4) result in undiscriminated binding to at least two sites located in the mitochondrial matrix: at one site, occupation by any of these three anions results in protection against uncoupling by K(+); at the second site, in the F(1)F(0)-ATPase, AsO(4) and SO(4) compete for binding against PO(4) leading to inhibition of the synthesis of ATP.

Arsenic resistance determinants from environmental bacteria.

Arsenic resistance determinants from 42 environmental bacterial isolates (32 Gram negative) were analyzed by DNA: DNA hybridization using probes derived from Escherichia coli and Staphylococcus plasmid or chromosomal arsenic resistance (ars) genes. In colony hybridization assays, 11 and 1 Gram negative strains hybridized with the E. coli chromosome and plasmid probes, respectively. No hybridization was detected using a probe containing only the arsA (ATPase) gene from E. coli plasmid or with a Staphylococcus plasmid ars probe. From Southern hybridization tests of some of the positive strains it was concluded that homology to ars chromosomal genes occurred within chromosome regions, except in an E. coli isolate where hybridization occurred in both the chromosome and a 130-kb plasmid. Our results show that DNA sequences homologous to E. coli ars chromosomal genes are commonly present in the chromosomes of environmental arsenic-resistant Gram negative isolates.

The mitochondrial membrane permeability transition induced by inorganic phosphate or inorganic arsenate. A comparative study.

The membrane permeability transition (MPT) induced by Ca2+ and Pi or Asi was studied in rat kidney mitochondria. Membrane potential, Ca2+ transport and swelling were used to monitor the MPT. Asi promoted a faster and more extensive collapse of membrane potential, Ca2+ release and swelling than Pi. The MPT induced by Pi was fully blocked by Mg(2+)+ADP, spermine+ADP, Mg(2+)+ cyclosporin A (CSA), and ADP+CSA. In contrast, the MPT induced by Asi was only prevented, although not completely, by CSA+Mg2+ or ADP+CSA. Asi, but not Pi, was able to cause collapse of membrane potential in the presence of Sr2+. Carboxyatractyloside (CAT) produced collapse of membrane potential at a lower concentration in the presence of Asi+Ca(2+)+ADP than with Pi+Ca(2+)+ADP. The addition of Pi+Ca2+ to [14C]-ADP loaded mitochondria brought about a greater ADP release than Asi+Ca2+. The ADP release was CAT-sensitive with Pi but it was only partially blocked by Asi. The diminution of external pH did not inhibit the MPT induced by Pi or Asi. The results of this study suggest that the adenine nucleotide translocase does not have an essential role in the MPT induced by Asi+Ca2+.

Effect of arsenic (V) on the antioxidant defense system: in vitro oxidation of rat plasma lipoprotein.

Adult male rats were treated orally with sodium arsenate (10 mg As/kd/day) for 2 days, and in increase in hepatic glutathione level was seen. Ascorbic acid content increased in both liver and plasma of intoxicated animals. Hepatic activities of superoxide dismutase and glutathione peroxidase did not change with the treatment and there was no increase in the level of lipid peroxidation measured as thiobarbituric acid-reacting substances (TBARS). Arsenic decreased the plasma level of uric acid and increased the plasma triglycerides content without modifying vitamin E levels. Both total lipoproteins and very low density lipoprotein plus low density lipoprotein (VLDL + LDL) fractions demonstrated greater propensity for in vitro oxidation than the corresponding untreated rats. The last finding might be a useful parameter for determining the degree of oxidative stress in the initial steps of intoxication with arsenic.

Leishmania (V.) guyanensis: isolation and characterization of glucantime-resistant cell lines.

A glucantime sensitive Leishmania (V.) guyanensis strain was used to obtain in vitro resistant cell lines, by increments in glucantime concentrations employing both one step and stepwise protocols. Whereas the effective concentration of drug that inhibited the growth of wild type cells by 50% (EC50 value) was 0.20 mg Sb(v)/mL, the resistant cells were able to grow in glucantime concentrations greater than 8.0 mg/mL. The resistant cell lines were partially characterized by their in vitro response to glucantime, the stability of resistance phenotype, cross resistance to a range of drugs, and also by the analysis of total DNA fragments generated by restriction endonucleases and blot hybridization. Amplified DNA sequence similar to a P-glycoprotein analog from Leishmania tarentolae (ltpgpA gene) was observed in all the resistant cell lines obtained through the one-step protocol. These cell lines showed cross resistance to heavy metals but were sensitive to puromycin, vinblastine, and pentostam.

Location of the beta-galactosidase of the yeast Kluyveromyces marxianus var. marxianus ATCC 10022.

During the growth of Kluyveromyces marxianus var. marxianus ATCC 10022 on lactose, peaks of glucose, but not beta-galactosidase activity, were detected in culture medium. Harvested and washed whole cells produced glucose and galactose from lactose, or ortho-nitro-phenol from the chromogenic substrate ortho-nitro-phenyl-beta-D-galactopyranoside (ONPG), indicating that beta-galactosidase is physically associated with cells. ONPG hydrolysis by whole cells presented a monophasic kinetics (Km 36.6 mM) in lactose exponential growth phase cells, but a biphasic kinetics (Km 0.2 and 36.6 mM) in stationary growth phase cells. Permeabilization with digitonin or disruption of cells from both growth phases led to monosite ONPG hydrolysis (Km 2.2 to 2.5 mM), indicating that beta-galactosidase is not located in the periplasm. In addition, the energy inhibitors fluoride or arsenate, as well as the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) prevented ONPG hydrolysis by whole cells. These findings indicate that energy coupled transmembrane transport is the rate-limiting step for intracellular ONPG cleavage. The taxonomic and physiologic implications of the exclusive intracellular location of beta-galactosidase of K. marxianus var. marxianus ATCC 10022 are discussed.

Effect of inorganic phosphate concentration on the nature of inner mitochondrial membrane alterations mediated by Ca2+ ions. A proposed model for phosphate-stimulated lipid peroxidation.

Addition of high concentrations (>1 mm) of inorganic phosphate (Pi) or arsenate to Ca2+-loaded mitochondria was followed by increased rates of H2O2 production, membrane lipid peroxidation, and swelling. Mitochondrial swelling was only partially prevented either by butylhydroxytoluene, an inhibitor of lipid peroxidation, or cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. This swelling was totally prevented by the simultaneous presence of these compounds. At lower Pi concentrations (1 mm), mitochondrial swelling is reversible and prevented by cyclosporin A, but not by butylhydroxytoluene. In any case (low or high phosphate concentration) exogenous catalase prevented mitochondrial swelling, suggesting that reactive oxygen species (ROS) participate in these mechanisms. Altogether, the data suggest that, at low Pi concentrations, membrane permeabilization is reversible and mediated by opening of the mitochondrial permeability transition pore, whereas at high Pi concentrations, membrane permeabilization is irreversible because lipid peroxidation also takes place. Under these conditions, lipid peroxidation is strongly inhibited by sorbate, a putative quencher of triplet carbonyl species. This suggests that high Pi or arsenate concentrations stimulate propagation of the peroxidative reactions initiated by mitochondrial-generated ROS because these anions are able to catalyze Cn-aldehyde tautomerization producing enols, which can be oxidized by hemeproteins to yield the lower Cn - 1-aldehyde in the triplet state. This proposition was also supported by experiments using a model system consisting of phosphatidylcholine/dicethylphosphate liposomes and the triplet acetone-generating system isobutanal/horseradish peroxidase, where phosphate and Ca2+ cooperate to increase the yield of thiobarbituric acid-reactive substances.

The Ca(2+)-ATPase isoforms of platelets are located in distinct functional Ca2+ pools and are uncoupled by a mechanism different from that of skeletal muscle Ca(2+)-ATPase.

Vesicles derived from the dense tubular system of platelets possess a Ca(2+)-ATPase that can use either ATP or acetyl phosphate as a substrate. In the presence of phosphate as a precipitating anion, the maximum amount of Ca2+ accumulated by the vesicles with the use of acetyl phosphate was only one-third of that accumulated with the use of ATP. Vesicles derived from the sarcoplasmic reticulum of skeletal muscle accumulated equal amounts of Ca2+ regardless of the substrate used. When acetyl phosphate was used in platelet vesicles, the transport of Ca2+ was inhibited by Na+, Li+, and K+; in sarcoplasmic reticulum vesicles, only Na+ caused inhibition. When ATP was used as substrate, the different monovalent cation had no effect on either sarcoplasmic reticulum or platelet vesicles. The catalytic cycle of the Ca(2+)-ATPase is reversed when a Ca2+ gradient is formed across the vesicle membrane. The stoichiometry between active Ca2+ efflux and ATP synthesis was one in platelet vesicles and two in sarcoplasmic reticulum vesicles. The coupling between ATP synthesis and Ca2+ efflux in sarcoplasmic reticulum vesicles was abolished by arsenate regardless of whether the vesicles were loaded with Ca2+ using acetyl phosphate or ATP. In platelets, uncoupling was observed only when the vesicles were loaded using acetyl phosphate. In both sarcoplasmic reticulum and platelet vesicles, the effect of arsenate was antagonized by thapsigargin (2 microM), micromolar Ca2+ concentrations, P(i) (5-20 mM), and MgATP (10-100 microM). Trifluoperazine also uncoupled the platelet Ca2+ pump but, different from arsenate, this drug was effective in vesicles that were loaded using either ATP or acetyl phosphate. Trifluoperazine enhanced Ca2+ efflux from both sarcoplasmic reticulum and platelet vesicles; thapsigargin, Ca2+, Mg2+, or K+ antagonized this effect in sarcoplasmic reticulum but not in platelet vesicles. The data indicate that the Ca(2+)-transport isoforms found in sarcoplasmic reticulum and in platelets have different kinetic properties.

Inorganic-ion resistance by bacteria isolated from a Mexico City freeway.

Bacteria were isolated from soil samples, containing high exchangeable lead concentrations, obtained from a busy freeway in the México City metropolitan area. Forty-five selected strains (86.7% Gram-positive) had a single MIC distribution pattern for lead (800-1600 micrograms/ml lead nitrate) and were considered lead-resistant. The isolates showed variable levels of resistance to arsenate (86.7%), chromate (66.7%), cadmium (57.6%), and mercury (31.1%) ions. Multiple inorganic-ion resistance was shown by all strains.

Molybdenum uptake in Escherichia coli K12.

Molybdenum uptake was examined in Escherichia coli K12 using the radionuclide 99Mo. The molybdenum uptake system was characterized in an unusual chlD strain, which appeared to be normal in uptake of the MoO4(2-) ion but altered in subsequent molybdenum processing. As a consequence, molybdenum could be chased from cells in the chlD strain, while it was irreversibly assimilated in the wild-type strain. Molybdenum uptake showed a biphasic kinetic curve, with a very rapid binding followed by a slow uptake phase. The uptake appeared to involve an active transport system. Molybdenum, probably in the form of molybdate, accumulated by a factor of about 30 in the cells. An energy source was necessary and uptake was inhibited by arsenate, but not by CCCP (carbonyl cyanide m-chlorophenylhydrazone). The uptake system saturated with a Km of 2.5-2.7 x 10(-8) M. Uptake seemed to depend on a periplasmic binding protein, since cold shock treatment and arsenate abolished uptake. A molybdate binding protein activity was detected in the periplasmic fluid with a KD of 9 nM. Sulphate inhibited uptake and the uptake activity was pH dependent, with an apparent pK of 6.7. These results imply that molybdate transport belongs to the family of energy-dependent periplasmic binding protein systems. An explanation for the peculiar behaviour of the chlD strain used in this work is proposed.

Effects of 2,4-dichlorophenoxyacetic acid on Rhizobium sp. growth and characterization of its transport.

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) produced a deleterious effect on the growth of Rhizobium sp. M4, that was reversed by transferring the cells to a fresh control medium. The uptake of 2,4-D by Rhizobium sp. was not affected by chloramphenicol, indicating that it is constitutive rather than inducible. The mechanism of transport also appears to be energy independent, since uptake was not inhibited by azide or arsenate.

D-glyceraldehyde-3-phosphate dehydrogenase from HeLa cells--1. Purification and properties of the enzyme.

1. D-GPDH from HeLa cells was isolated and purified. 2. Some basic kinetic constants are reported. 3. Sodium dodecyl polyacrylamide gel electrophoresis gave a single band with a molecular weight of approximately 36 K. 4. ATP and NADH inhibit competitively enzyme activity. 5. Comparative catalytic properties of GPDH from normal and tumor cells were effectuated.

Phosphate transport in isolated rat inner medullary collecting duct.

Phosphate transport by the inner medullary collecting duct of normal rats was studied using an in vitro microperfusion technique. Net (Jnet), lumen-to-bath (Jlb) and bath-to-lumen (Jbl) phosphate fluxes were measured using 32PO4 as tracer, in the absence of net water absorption. A net absorption of phosphate (22.3 +/- 3.3 pmol cm-2 s-1) was observed by direct determination, and was similar to the difference between the Jlb and Jbl (57.7 +/- 8.2 and 32.2 +/- 1.5 pmol cm-2 s-1 respectively). The addition of amiloride (10 microM) to the perfusate did not change the Jlb of phosphate but blocked the efflux of sodium. Also, the withdrawal of sodium from the bath and perfusion solution did not change the Jlb of phosphate. In parallel, the addition of ouabain (10 mM) to the bath fluid decreased the Jlb of sodium more (37%) than the Jlb of phosphate (12%) and did not change the Jbl of phosphate. The addition of arsenate (10 microM) to the perfusate both in the presence and in the absence of sodium caused a decrease in Jlb, but Jbl remained unchanged, and parathyroid hormone (10 U) added to the bath did not change the Jlb. The increase in pH of the bath and perfusion fluid was associated with an increase in the Jlb of phosphate, and the decrease in pH was similarly followed by a decrease in phosphate efflux. The Jbl did not change with the pH alterations. These data demonstrate that a net phosphate absorption takes place in rat inner medullary collecting duct perfused in vitro and that this transport appears to be independent of sodium absorption and the action of parathyroid hormone. Moreover, a decrease in luminal and bath pH induces a decrease in phosphate efflux.

Arsenate-induced fluorescence changes in the Ca(2+)-ATPase of sarcoplasmic reticulum membranes.

Arsenate, an analogue of inorganic phosphate, causes an increase in the intrinsic fluorescence of the Ca(2+)-ATPase of sarcoplasmic reticulum membranes. This increase in fluorescence is observed regardless of whether Ca(2+)-loaded or leaky vesicles are assayed. The maximal fluorescence change (2-3%) is observed at pH 6.0 in the presence of Mg2+ and is abolished by the addition of micromolar Ca2+ concentrations. Dimethyl sulfoxide (20% v/v) increases the enzyme's affinity for arsenate one order of magnitude. It is concluded that arsenate, after binding, promotes the same conformational change of the enzyme as that produced by Pi.