μM HgCl2 Research Articles

HEAVY‐METAL INDUCED CHANGES IN NONPROTEINACEOUS THIOL LEVELS AND HEAVY‐METAL BINDING PEPTIDE IN TETRASELMIS TETRATHELE (PRASINOPHYCEAE)

The effects of mercury and cadmium on the intracellular level of nonproteinaceous thiols in a unicellular green alga Tetraselmis tetrathele (West) Butcher (Prasinophyceae) were investigated by using a fluorescent dye, 5‐chloromethylfluorescein (5CMF), as a probe for nonproteinaceous thiols. The 5CMF fluorescence was observed in cytoplasm, and the intensity of the fluorescence was decreased by exposure of the cells to HgCl2. Analysis of the fluorescent intensity of 5CMF by flow cytometry made it possible to distinguish cells in three states during the dying process caused by HgCl2: a normal state, a thiol‐depleted state, and a dead state. Depletion of nonproteinaceous thiols began within 30 min, and they were completely depleted at 2 h. Most cells died after 24 h of exposure to more than 3.0 μM HgCl2, whereas exposure up to 1.0 mM CdCl2 did not cause depletion of nonproteinaceous thiols or cell death within 48 h. HPLC analyses revealed that glutathione was a major nonprotein thiol in T. tetrathele and that it was oxidized by exposing the cells to HgCl2. Phytochelatins, which play a great role in the tolerance to heavy metals of higher plants and many algae, could not be found in T. tetrathele. However, a tripeptide, Arg‐Arg‐Glu, was found to be abundant, and it showed ability to bind Hg2+, suggesting that it functions to scavenge heavy metals as well as thiol molecules.

Inhibition of water channels by HgCl2 in intact wheat root cells

To assess the extent of water flow through channels in the membranes of intact higher plant cells, the effects of HgCl2 on hydraulic conductivity (LP) of wheat (Triticum aestivum L.) root cells were investigated using a pressure probe. The LP of root cells was reduced by 75% in the presence of 100 &mgr;M HgCl2. The K+-channel blocker tetraethylammonium had no effect on the LP at concentrations that normally block K+ channels. HgCl2 rapidly depolarized the membrane potential (Vm) of the root cells. The dose-response relationship of inhibition of LP and depolarization of Vm were not significantly different, with half-maximal inhibition occurring at 4. 6 and 7.8 &mgr;M, respectively. The inhibition of LP and the depolarization of Vm caused by HgCl2 were partially reversed by beta-mercaptoethanol. The inhibition of LP by HgCl2 was similar in magnitude to that caused by hypoxia, and the addition of HgCl2 to hypoxia-treated cells did not result in further inhibition. We compared the LP of intact cells with that predicted from a model of cortical cells incorporating water flow across both the plasma membrane and the tonoplast using measured values of water permeability from isolated membranes, and found that HgCl2 has other effects in addition to the direct inhibition of water channels.

Mercuric-Ion-Induced Gene Expression in Arabidopsis thaliana

ABSTRACT Fourteen ecotypes of Arabidopsis thaliana were grown on agar, containing up to 40 μM HgCl2. Two groups could be identified: (1) sensitive ecotypes growing only up to 30 μM and (2) tolerant ecotypes growing at 40 μM. The tolerant ecotypes accumulated up to 100 mg mercury/kg fresh weight in their rosettes. Arabidopsis thaliana wild-type Columbia (Col-wt) was analyzed in more detail and showed an exponential mercury accumulation with increasing Hg2+-concentrations in the medium. Increasing Hg2+-concentrations resulted in a delayed growth of the plants. The roundish shape of the leaves changed to a more elongated form, and the leaf color changed from dark green to bright green. Hg2+-induced transcripts of several genes known to be induced by oxidative stress were measured and compared with the induction by ozone and UV-B. Glutathione-S-transferase (GST1) and several pathogenesis-related protein (PR) mRNAs were not induced in plants grown on Hg2+-containing medium. However, ozone fumigation increased the level of all these transcripts transiently. Similarly, UV-B irradiation resulted only in a weak mRNA increase. Metallothionein 1a (MT1a) transcript accumulation increased with rising Hg2+-concentrations, whereas MT2a mRNA level was unchanged. In contrast, MT2a transcripts were induced by ozone, whereas MT1a transcripts were unaffected. UV-B irradiation had a weak effect on MT1a and no effect on MT2a transcripts. These results indicate that different abiotic stressors led to differential transcript changes of Arabidopsis thaliana.

Amelioration of lead and mercury effects on germination and rice seedling growth by antioxidants

Germination of two rice (Oryza sativa L.) cultivars (Ratna and IR 36) in the presence of 10 µM PbCl2 and 10 µM HgCl2 decreased germination percentage, germination index, shoot/root length, tolerance index and dry mass of shoots and roots. Mercury was more toxic than lead. Reduced glutathione (GSH), cysteine (Cys), ascorbic acid, and α-tocopherol alleviated the adverse effects of these metals on plants in the order GSH > Cys > ascorbic acid > α-tocopherol. The effects were more pronounced in tolerant cultivar IR 36 than in the relatively susceptible cultivar Ratna.

Strychnine and Glycine Protect Renal Proximal Tubules from Various Nephrotoxicants and Act in the Late Phase of Necrotic Cell Injury

We have previously shown that strychnine mimics the cytoprotective properties of glycine in renal proximal tubule (RPT) suspensions exposed to antimycin A (AA). The aims of this study were to determine whether the cytoprotective properties of strychnine applied to various types of nephrotoxicants and to examine the temporal aspects of the cytoprotection of glycine and strychnine. Tubular release of LDH activity was used as a marker of cell death. Glycine (2 mM) or strychnine (1 mM) added 5 min prior to the toxicant decreased LDH release in rabbit RPT suspensions exposed to 25 μM tetrafluoroethyl-L-cysteine (TFEC), 10 μM HgCl2, 0.5 mM t-butyl hydroperoxide (TBHP), or 0.2 mM bromohydroquinone (BHQ) for 4 hr, or 2 mM sodium cyanide (NaCN) for 2 hr. The relative rank order of effectiveness of glycine and strychnine was NaCN = TFEC > BHQ > DCVC ⪢ TBHP > HgCl2. The temporal aspects of strychnine and glycine protection were examined by exposing RPT to either AA or TFEC for 1 or 3 hr, respectively, and then adding either 1 mM glycine or 1 mM strychnine. Glycine and strychnine decreased LDH release in AA-treated RPT at 1.25 and 2 hr and TFEC-treated RPT at 4 hr. In addition, when RPT exposed to AA or TFEC and treated with strychnine or glycine were washed at either 1 or 4 hr, protection was eliminated at later time points. When glycine was added to RPT treated with either PCBC, TFEC, or DCVC 5 min prior to or 30, 60, 120, and 180 min following toxicant addition, LDH release was reduced at all time points. These results demonstrate that strychnine and glycine protect RPT from a variety of diverse nephrotoxicants, strychnine and glycine do not need to be present at the time of toxic insult, strychnine and glycine cytoprotection is reversible, and strychnine and glycine act in the late phase of necrotic cell injury.

HgCI<sub>2</sub>-lnduced Ion Transport Pathways in Ehrlich Ascites Tumor Cells

HgCl2 (40 µM) initially activates an anion-independent unidirectional Na+ influx in Ehrlich ascites tumor cells, which amounts to 61 ± 7 µmol·g dry wt-1 min-1 (± SEM, n = 5). The unidirectional Na+ influx in control cells in 13 ± 2 µmol·dry wt-1 • min-1 (± SEM, n = 5). The HgCl2-activated Na+ influx is slightly inhibited by amiloride (inhibitor of Na+/H+ exchange), but almost completely blocked by the anion-exchange inhibitor DIDS (4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid). HgCl2 also causes DIDS-sensitive cytoplasmic acidification followed by DIDS-sensitive cytoplasmic alkalinization. After addition of HgCl2 the cells initially shrink, whereupon they swell again. Half-maximal effect of HgCl2 is obtained with about 0.5 and 1 µM HgCl2 on cell swelling and cell shrinkage, respectively. The volume responses are both reduced in the presence of DIDS. The unidirectional K+ and Cl– effluxes are greatly stimulated by HgCl2. The stimulation of K+ efflux results in a Cl--dependent net loss of K+. In Cl– medium the K+ loss is 84 ± 11 µmol·g dry wt–1 • min–1 ( ± SEM, n = 6). In NO–3 medium, it is reduced to 24 ± 6 µmol·g dry wt-1 • min-1 ( ± SEM, n = 4). The HgCl2 activated K+ efflux in Cl– medium is reduced to 21 ± 6 µmol·g dry wt–1 • min–1 ( ± SEM, n = 3) by DIDS. Finally, an instantaneous hyperpolarization of the cell membrane potential was observed after addition of HgCl2. It is concluded that HgCl2 in Ehrlich cells activates (i) a DIDS-sensitive, Na+-dependent anion exchange system; (ii) a DIDS-sensitive and anion-dependent K+ transport system, and (iii) K+ channels.

Effectiveness of various treatments in retarding microbial activity in sediment trap material and their effects on the collection of swimmers

Laboratory and field experiments were conducted to determine the effectiveness of various poison, preservative, and antibiotic treatments currently in common use in sediment trap work. Our results agree with previous reports that most biological activity in sediment traps can be inhibited by appropriate concentrations of formaldehyde, chloroform, or mercuric chloride. Sodium azide appears to be less effective at inhibiting biological activity, while antibiotics and salt alone are probably too ineffective for long‐term deployments. On the basis of laboratory experiments, effective concentrations when sustained throughout the deployment are 50% saturated chloroform, 37 mM formaldehyde, or 180 µM HgCl2. Our results also indicate, however, that the weight of swimmers (zooplankton which swim into the trap and die there from contact with the poison) collected in sediment traps is higher when more effective treatments are used. The presence of large numbers of swimmers can greatly affect the flux and composition of particulate organic matter collected in sediment traps and may decrease the effectiveness of the various treatments in inhibiting microbial activity in the traps.

Degradation of organomercurials in Bacillus cereus.

Degradation of phenylmercuric acetate (PMA) and p-chloromercuribenzoic acid (PCMB) by cells of a mercury resistant strain of Bacillus cereus was demonstrated. Degradation of PMA was also demonstrated with the crude extract of the B. cereus cells grown in nutrient broth containing 1 μm PMA or 10 μm HgCl2. The crude extract seems to split the carbon-mercury bond of PMA and form benzene and the mercuric ion as degradation products. The splitting enzyme was separated from mercuric reductase which catalyzes the reduction of the mercuric ion by gel filtration on Sephadex G 100.

The regulation of ionic nickel uptake and cytotoxicity by specific amino acids and serum components

The effects of serum components and amino acids on the uptake and cytotoxicity of NiCl2 were examined in cultured Chinese hamster ovary (CHO) cells. CHO cells maintained in a minimal salts/glucose medium accumulated 10-fold more(63)Ni than did cells maintained in complete medium supplemented with 10% fetal bovine serum. Cell-surface binding of(63)Ni appeared to account for the majority of this increased accumulation of cell-associated nickel observed in the simple maintenance medium since such increases were reduced 70% by trypsin treatment. The addition of the Ni(2+)-binding amino acids cysteine or histidine to the salts/glucose medium markedly decreased(63)Ni accumulations, an effect not observed following addition of any of several amino acids that do not bind Ni(2+). Supplementation of the salts/glucose medium with fetal bovine serum decreased in a concentration dependent fashion both the(63)Ni(2+) uptake and cell detachment caused by Ni(2+), while dialyzed (amino acid-free) serum was 3-5-fold less effective than undialyzed serum at reducing(63)Ni(2+) uptake and similarly exhibited only a slight protective effect against nickel-induced cytotoxicity. Supplementation of dialyzed serum with cysteine at levels approximating those in whole serum partially restored its inhibitory activity toward nickel uptake by cells and restored completely its inhibition of nickel's cytotoxicity, indicating the predominant role of specific amino acids over serum proteins in regulating the uptake and subsequent cytotoxicity of Ni(2+). Addition of cysteine to the salts/glucose medium during a 2 h exposure of cells to either 100 μM HgCl2 or 1 mM NiCl2 masked the cytotoxic effects of these metal ions. These results demonstrate the importance of extracellular small molecular weight metal ion chelators in altering the biological effects of metal ions at the level of metal uptake.

Similarity in the acute cytotoxic response of mammalian cells to mercury (II) and X-rays: DNA damage and glutathione depletion

Pronounced strand breakage of DNA analyzed by alkaline elution techniques was produced in intact Chinese hamster ovary cells by 25 μM HgCl2 within 1 hr or 100 μM HgCl2 within 15 min. HgCl2-induced strand breakage was directly proportional to concentration up to 100 μM and to time within 1 hr. Levels of reduced glutathione decreased following HgCl2 in parallel with the induction of DNA strand breakage. Evidence is presented that this rapid and pronounced induction of DNA strand breaks and other cytotoxic responses following acute exposure to HgCl2 resembles the cellular effects of X-rays.

Properties of four molecular forms of N-acetyl-beta-D-hexosaminidase isolated from germinating seeds of fenugreek (Trigonella foenum graecum).

Four molecular forms of N-acetyl-β-d-hexosaminidase located in different parts of the germinating fenugreek seeds have been characterized and isolated. Their activities vary during the germination process. The optimal pH and temperature as well as Km value towards synthetic substrates were found to be different for the N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities of each molecular form. Both N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities of molecular form I increased to a similar extent in the presence of Fe2+ ions whereas both activities of molecular forms II, III and IV were depressed. In the presence of Fe3+ ions, only the N-acetyl-β-d-galactosaminidase activity of molecular form I increased. No inhibition of either activity of N-acetyl-hexosaminidase form I was obtained in the presence of 5 μM HgCl2 or p-chloromercuribenzoate, whereas both activities of the other forms were inhibited. These four N-acetyl-hexosaminidases hydrolyse synthetic and natural substrates: hexosaminidase form I liberates the 1,4-β-linked N-acetyl-glucosamine residue, form II liberates the 1,6-linked residue at a very slow rate, whereas forms III and IV liberate 1,2-β-linked N-acetylglucosamine. On isoelectric focusing, pI values of 6.78, 6.3, 4.9 and 4.65 were determined for the forms I, II, III and IV respectively. Using Sepharose 6B filtration, molecular weights were estimated to be 84 000 ± 10 000, 72 000 ± 10 000, 180 000 ± 10 000 and 150 000 ± 10 000 for the forms I, II, III and IV respectively. Treatment with dodecyl sulfate resulted in the dissociation of all four forms with bands having a molecular weight of 28 000 ± 2000, 24 000 ± 2000, 30 500 ± 2000 and 30 000 ± 2000 for the forms I, II, III and IV respectively. These results suggest that the four hexosaminidases may be polymers formed by association of between three and six subunits of identical molecular weight. Immunological studies reveal common antigenic determinants between forms I and II located in cotyledon and endosperm and between forms III and IV located predominantly in the embryo.

The Effect of Sub-Lethal Concentrations of Mercury and Zinc on Chlorella

Summary Chlorella cultures maintained continuously in replenished solutions of heavy metals (1 mM ZnCl2, 1 μM HgCl2 or 0.1 μm phenyl mercuric acetate) initially exhibit symptoms of metal toxicity, which are characterised by a sharp reduction in the pigment content and in the rates of cell division and metabolic activity. There is a concurrent gain in the net carbon balance of the culture due to the inhibition of export of photosynthates from the cells by heavy metals ( De Filippis and Pallaghy, 1976a , De Filippis and Pallaghy, 1976b ). After a period of 40 days (approximating 40 series of cell division) the cultures develop resistance and almost fully regain the rates of cell division displayed by control cultures. In attempting to elucidate the mechanism of resistance, we studied the fate of the heavy metals in cultures of sensitive and resistant cells. Evidence from 65Zn uptake studies indicate that resistance is accompanied by an inhibition of the temperature sensitive component of zinc uptake and by a reduction in the number of exchange sites available for zinc in the free space (cell walls) per unit dry weight of the cells. Tolerance is therefore characterised by the development of a typical exclusion mechanism. Mercury resistance is primarily associated with an increased capacity for resistant cells to volatilize mercury out of solution by enzymic means. The ethylene generated by metal stressed cultures (up to 1.3 pmol/mg dry wt/hr) is sufficient to account for approximately 25%, and a mercury-reducing enzyme system for the remainder of the mercury volatilized.

Purification and Properties of a Glycohydrolase from Calf Thymus Splitting Ribose-Ribose Linkages of Poly(Adenosine Diphosphate Ribose)

Abstract A new enzyme splitting the ribose-ribose bonds of poly-(adenosine diphosphate ribose) was purified 200-fold from calf thymus. The molecular weight of the enzyme was estimated to be 48,000 by gel filtration and 53,000 by sucrose density gradient centrifugation. Its optimum pH was around 7.5. For full activity 10 mm 2-mercaptoethanol or dithiothreitol was required. Activity was inhibited considerably by 10 µm p-chloromercuriphenylsulfonate or HgCl2. The Km value for poly(adenosine diphosphate ribose) was 0.58 µm. Adenosine cyclic 3' : 5'-monophosphate and adenosine diphosphate ribose inhibited the reaction, causing 50% inhibition at concentrations of 0.3 mm and 1.1 mm, respectively. Calf thymus histone f2a, f2b, and f3, protamine, and poly-l-lysine inhibited the activity. The inhibition by histone f2a was reversed by DNA. The enzyme hydrolyzed poly(adenosine diphosphate ribose) in a fashion of an exoglycosidase. The end product of hydrolysis is adenosine diphosphate ribose. This enzyme may function in regulation of the chain length of poly(adenosine diphosphate ribose), which was previously claimed to play a role in DNA synthesis or in the structure of chromatin.

Purification and Properties of Nicotinamide Deamidase from Flavobacterium peregrinum

Abstract Nicotinamide deamidase from Flavobacterium peregrinum was purified about 210-fold with the use of protamine and MnCl2 treatments, acetone fractionation, ammonium sulfate fractionation, Sephadex G-200, and Cm-Sephadex column chromatography. The optimum pH of the purified nicotinamide deamidase is 6.5, and the Km value for nicotinamide is 0.2 µm. The molecular weight was estimated by means of gel filtration to be 48,000. The enzyme activity of the crude preparation was increased by 2.5-fold by addition of Mn2+ at a concentration of 10 mm followed by incubation at 37° for 30 min. The purified enzyme preparation was inactivated by dialysis in the absence of Mn2+ and no recovery of enzyme activity was possible even when Mn2+ or Mn2+ plus cysteine was added to the reaction mixture. When the enzyme was dialyzed overnight in the presence of Mn2+, almost all of the enzyme activity was retained. A dialysis of the enzyme against 0.01 m maleate buffer (pH 6.5) containing 5 µm HgCl2 resulted in complete retention of enzyme activity when 50 mm cysteine was added to the reaction mixture. CuSO4 and SnCl2 also showed effects similar to that of HgCl2, but their potency for enzyme stability was small. Mg2+, Ni2+, Zn2+, and Fe2+ did not show any effect on enzyme activity. All the nicotinamide analogues having a trivalent nitrogen in the pyridine ring showed a competitive inhibition of nicotinamide deamidation, whereas compounds having a tetravalent nitrogen seemed to have no effect. From these results the structure of the enzyme-metal-substrate complex was proposed as [see PDF for equation]