Cytotoxicity and genotoxicity of low doses of mercury chloride and methylmercury chloride on human lymphocytes in vitro

Mercury is a xenobiotic metal that is a highly deleterious environmental pollutant. The biotransformation of mercury chloride (HgCl2) into methylmercury chloride (CH3HgCl) in aquatic environments is well-known and humans are exposed by consumption of contaminated fish, shellfish and algae. The objective of the present study was to determine the changes induced in vitro by two mercury compounds (HgCl2 and CH3HgCl) in cultured human lymphocytes. Short-term human leukocyte cultures from 10 healthy donors (5 females and 5 males) were set-up by adding drops of whole blood in complete medium. Cultures were separately and simultaneously treated with low doses (0.1 to 1000 µg/l) of HgCl2 and CH3HgCl and incubated at 37ºC for 48 h. Genotoxicity was assessed by chromosome aberrations and polyploid cells. Mitotic index was used as a measure of cytotoxicity. A significant increase (P < 0.05) in the relative frequency of chromosome aberrations was observed for all concentrations of CH3HgCl when compared to control, whether alone or in an evident sinergistic combination with HgCl2. The frequency of polyploid cells was also significantly increased (P < 0.05) when compared to control after exposure to all concentrations of CH3HgCl alone or in combination with HgCl2. CH3HgCl significantly decreased (P < 0.05) the mitotic index at 100 and 1000 µg/l alone, and at 1, 10, 100, and 1000 µg/l when combined with HgCl2, showing a synergistic cytotoxic effect. Our data showed that low concentrations of CH3HgCl might be cytotoxic/genotoxic. Such effects may indicate early cellular changes with possible biological consequences and should be considered in the preliminary evaluation of the risks of populations exposed in vivo to low doses of mercury.

Genotoxicity of inorganic mercury salts based on disturbed microtubule function

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 μM, and inhibition is complete at about 10 μM. In this range, the tubulin assembly is fully (up to 6 μM) or partially (∼6-10 μM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect- concentration for inhibition of microtubule assembly in vitro was 1 μM Hg2+, the IC50 5.8 μM. Mercury(II) salts at the IC 50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a "gliding assay" mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 μM and a complete inhibition is reached at 1 μM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 μM HgCl2. Between 15 and 20 μM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 μM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations (100 μM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg 2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

Genotoxicity of inorganic mercury salts based on disturbed microtubule function

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 muM, and inhibition is complete at about 10 muM. In this range, the tubulin assembly is fully ( up to 6 muM) or partially (similar to 6 - 10 muM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect-concentration for inhibition of microtubule assembly in vitro was 1 muM Hg2+, the IC50 5.8 muM. Mercury(II) salts at the IC50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a gliding assay'' mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 muM and a complete inhibition is reached at 1 muM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 muM HgCl2. Between 15 and 20 muM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 muM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations ( 100 muM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

A preliminary characterization of mercury uptake by the sulfate-reducing bacteria Desulfovibrio desulfuricans

The focus of this research is to determine if a relationship exists between the stability constant and the initial uptake rate of a mercury species by bacteria. Cultures of the sulfate-reducing bacteria (SRB) strain Desulfovibrio desulfuricans G20 were washed with a bicarbonate buffer solution containing either lactate and sulfate or pyruvate and fumarate. The washed cell solutions were then spiked with either mercury bound to natural organic matter (Hg-NOM) or neutral mercury chloride (HgCl2), followed by sampling over time to provide kinetic data. Despite the significantly different stability constants for Hg-NOM and HgCl2, the calculated initial rate constants for mercury uptake for these two types of complexes appeared to be comparable. Uptake of mercury sulfide species was inconclusive due to possible formation of cinnabar. A simple model that is based on assumptions of passive diffusion and facilitated uptake of mercury by bacteria was evaluated for its potential to simulate the uptake. The model results only agreed with experimental data for HgCl2 uptake.

Interaction of metal salts with cytoskeletal motor protein systems

Interactions of chemicals with the microtubular network of cells may lead to genotoxicity. Micronuclei (MN) might be caused by interaction of metals with tubulin and/or kinesin. The genotoxic effects of inorganic lead and mercury salts were studied using the MN assay and the CREST analysis in V79 Chinese hamster fibroblasts. Effects on the functional activity of motor protein systems were examined by measurement of tubulin assembly and kinesin-driven motility. Lead and mercury salts induced MN dose-dependently. The no-effect-concentration for MN induction was 1.1 μM PbCl2, 0.05 μM Pb(OAc)2 and 0.01 μM HgCl2. The in vitro results obtained for PbCl2 correspond to reported MN induction in workers occupationally exposed to lead, starting at 1.2 μM Hg(II) (Vaglenov et al., 2001, Environ. Health Perspect. 109, 295-298). The CREST Analysis indicate aneugenic effects of Pb(II) and aneugenic and additionally clastogenic effects of Hg(II). Lead (chloride, acetate, and nitrate) and mercury (chloride and nitrate) interfered dose-dependently with tubulin assembly in vitro. The no-effect-concentration for lead salts in this assay was 10 μM. Inhibition of tubulin assembly by mercury started at 2 μM. The gliding velocity of microtubules along immobilised kinesin molecules was affected by 25 μM Pb(NO3)2 and 0.1 μM HgCl2 in a dose-dependent manner. Our data support the hypothesis that lead and mercury genotoxicity may result, at least in part, via disturbance of chromosome segregation via interaction with cytoskeletal proteins.

Etablierung neuer Methoden zur in vitro Kultivierung der Baumart Fraxinus excelsior L. und Entwicklung von Verfahren zur Kryokonservierung von in vitro Sprossspitzen

Die hier vorliegende Arbeit wurde im Rahmen eines europäischen Projektes mit dem Titel „Improving Fraxinus (Ash) productivity for European needs by testing, selection, propagation and promotion of improved genetic resources“ an der Niedersächsischen Forstlichen Versuchsanstalt, Abteilung Waldgenressourcen erstellt. Im Rahmen des Projektes wurden 62 Plusbäume aus einem 15 Jahre alten europäischen Herkunfts-/ Nachkommenschaftsversuch in den Niedersächsischen Forstämtern Bovenden und Dannenberg nach den Kriterien Stammform und Wuchsleistung für die vegetative Vermehrung ausgewählt. Ziel dieser Arbeit war die Optimierung bestehender in vitro Protokolle sowie die Entwicklung eines bisher noch nicht existierenden Kryokonservierungsprotokolls für in vitro Sprossspitzen. Im ersten Teil dieser Arbeit wird die Entwicklung des in vitro Protokolls für Fraxinus excelsior dargestellt. Die Optimierung der Methoden zur Etablierung, Vermehrung und Bewurzelung erfolgte durch Versuchsreihen mit unterschiedlichen Klonen, so dass insgesamt 26 der selektierten Plusbäume erfolgreich in vitro etabliert werden konnten. Achselknospen frischer Triebe der Pfropflinge der Mutterbäume stellten die beste Explantatquelle dar. Die Explantate wurden mit 0,2 % Quecksilberchlorid (HgCl2) oberflächensterilisiert bevor sie auf hormonfreies Woody Plant Medium (WPM) transferiert wurden. Nach zwei Wochen erfolgte ein Transfer auf WPM mit 4 mg/l 6-Benzylaminopurine (BAP) und 0,15 mg/l Indole-3-butyric acid (IBA). Die besten Vermehrungsraten wurden auf WPM mit 4 mg/l BAP, 0,15 mg/l IBA und 0,01 mg/l TDZ und 0,7 % Agar in Honiggläsern mit einem Plastikdeckel erzielt. Als Bewurzelungsmedium wurde 0,5 konzentriertes Murashige und Skoog (MS) Medium mit 2 mg/l IBA, 0,25 mg/l BAP und 0,8 % Agar verwandt. Im zweiten Teil der Arbeit werden die Versuchsreihen zur Entwicklung des Kryokonservierungsprotokolls von in vitro Sprossspitzen dargestellt. Zur Entwicklung der Methode wurden die Vorbehandlungsbedingungen verbessert und zwei Techniken, die Alginat- / Dehydrati-onsmethode und die Vitrifikationsmethode mit Hilfe der sogenannten PVS2-Lösung (Plant Vitrification solution number 2) getestet. Die optimierte PVS2-Methode erwies sich als die für Esche besser geeignete Technik und ließ sich erfolgreich zur Kryokonservierung juveniler und adulter Kulturen anwenden. Die Regenerationsraten lagen zwischen 50 und 100 % für juvenile bzw. 50 und 80 % für adulte Kulturen.

Atividade β-D-N-Acetilglucosaminidásica de enzimas imobilizadas extraídas da Artemia franciscana e possíveis aplicações biotecnológicas

A β-D-N-acetilglucosaminidase extracted and partially isolated from crustacean Artemia franciscana by ammonium sulfate precipitation and filtration gel chromatography Bio Gel A 1.5m. the enzyme was immobilized on ferromagnetic Dacron yielding a insoluble active derivative with 5.0 units/mg protein and 10.35% of the soluble enzyme activity. β-D-N-acetilglucosaminidase-ferromagnetic Dacron was easily removed from the reaction mixture by a magnetic field, it was reused for ten times without loss in its activity. The ferromagnetic Dacron was better activated at pH 5.0. The particles visualized at scanning electron microscope (SEM) had presented different sizes, varying between 721nm and 100µm. Infra red confirmed immobilization on support, as showed by primary amino peaks at 1640 and 1560 cm-1 . The immobilize enzyme presented Km of 2.32 ± 0.48 mM and optimum temperature of 50°C. Bought presented the same thermal stable of the soluble enzyme and larger enzymatic activity at pH 5.5. β-D-N-acetilglucosaminidase-Dacron ferromagnético showed sensible for some íons as the silver (AgNO3), with loss of activity. The β-D-N acetilglucosaminidase activity for mercury chloride (HgCl2), whom is one of the most toxic substance joined in nature, it was presented activity already diminished at 0,01mM and lost total activity at 4mM, indicating sensitivity for this type of metal. β-D-N-acetilglucosaminidase-ferromagnetic Dacron showed degradative capacity on heparan sulfate, the enzyme still demonstrated degradative capacity on heparan sulphate, suggesting a possible application to produce fractions of this glycosaminoglycan

Purificação e caracterização de uma ß-N-acetillhexosaminadase extraída do mamífero marinho Sotalia fluviatilis

This report shows 2232 times purification of a βNAcetylhexosaminidase from hepatic extracts from the sea mammal Sotalia fluviatilis homogenate with final recovery of 8,4%. Sequenced steps were utilized for enzyme purification: ammonium sulfate fractionation, Biogel A 1.5 m, chitin, DEAESepharose and hydroxyapatite chromatographies. The protein molecular mass was estimated in 10 kDa using SDSPAGE and confirmed by MALDITOF. It was found to have an optimal pH of 5.0 and a temperature of 60°C. Using pnitrophenylNAcetylβDglycosaminide apparent Km and Vmax values were of 2.72 mM and 0.572 nmol/mg/min, respectively. The enzyme was inhibited by mercury chloride (HgCl2) and sodium dodecil sulfate (SDS)

Esterase patterns in species of the triatome bug Rhodnius

The aim of the present study was to analyse esterase patterns in three triatomine species of Rhodnius genus. Four loci, Est 1, Est 2, Est 3 and Est 4, were found. The corresponding enzymes were characterized as carboxylesterases (E.C. 3.1.1.1) or cholinesterases (E.C. 3.1.1.8) based on inhibitory experiments, using eserine sulphate, malathion, mercury chloride, p-chloromercuribenzoate (pCMB) and iodoacetamide. Low genetic variability was observed: Est 1, Est 2 and Est 3 were monomorphic in Rhodnius domesticus, Rhodnius robustus and Rhodnius neivai, whereas locus Est 4 was polymorphic in the first two species. The UPGMA analysis based on esterase genotypic frequencies indicated greater similarity between R. domesticus and R. robustus when compared with R. neivai. The present study expands our knowledge about genetic variability among triatomines and accords with the hypothesis that R. domesticus is a species derived from R. robustus.

Variações bioquímicas em Pterygoplichthys anisitsi e Oreochromis niloticus (Pisces: Teleostei) após exposição a cloreto de mercúrio

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação neurocomportamental da exposição crônica ao Mercúrio inorgânico na memória social e memória emocional de ratos wistar machos adultos

O mercúrio inorgânico é facilmente absorvido por ingestão ou via cutânea. Entretanto, uma quantidade relativamente pequena de Hg²⁺ atravessa a barreira hematoencefálica ou as membranas biológicas, sendo em ratos adultos, o transporte axonal retrógrado a única via para a absorção de Hg²⁺ por neurônios, apresentando um forte potencial neurotóxico. Desta forma, o presente estudo objetivou investigar os efeitos da exposição crônica ao cloreto de mercúrio em memória social e emocional de ratos adultos. Para isso utilizou-se ratos Wistar, machos (n=40), com 5 meses de idade, distribuídos em dois grupos, um dos quais foi exposto ao Cloreto de Mercúrio (HgCl₂) via oral, por gavagem intra-gástrica (0,375mg/Kg), durante 45 dias. O outro grupo, denominado grupo controle (n=20) recebeu água destilada por gavagem. Foram utilizados os seguintes testes comportamentais: teste do campo aberto, teste de reconhecimento social para avaliação de memória social; o Teste do Labirinto em T Elevado (LTE) foi usado para avaliar o aprendizado do estado de esquiva e as memórias de curta e longa-duração. Após a finalização dos testes, os animais foram sacrificados para a dosagem do mercúrio total no hipocampo e através de um Espectrofotômetro de Absorção Atômica. Os resultados revelaram que os animais submetidos à exposição ao cloreto de mercúrio não manifestaram déficits em atividade exploratória. Nos dados do Teste de Reconhecimento Social, observamos que não houve alteração em memória social. No teste do LTE, o grupo exposto ao HgCl₂ necessitou de um número maior de exposições para aquisição do critério de esquiva (p<0,05) e apresentaram latência maior no braço aberto do aparato (p<0,05). Após 24 horas, verificou-se que os animais expostos passaram menos tempo no braço fechado em relação ao grupo controle, sugerindo déficits de memória de longa duração. Ao observar apenas o grupo HgCl₂, percebeu-se uma melhora no reteste, indicando preservação na memória de curta duração. Os dados de espectrometria de absorção atômica mostraram uma maior deposição de mercúrio no hipocampo de animais intoxicados, em relação aos animais do grupo controle.

Pore water geochemistry of Håkon Mosby mud volcano sediments measured at station PS74/169-1_PUC-15 (Z1-S1, new flow surface)

Pore water and turnover rates were determined for surface sediment cores obtained in 2009 and 2010. The pore water was extracted with Rhizons (Rhizon CSS: length 5 cm, pore diameter 0.15 µm; Rhizosphere Research Products, Wageningen, Netherlands) in 1 cm-resolution and immediately fixed in 5% zinc acetate (ZnAc) solution for sulfate, and sulfide analyses. The samples were diluted, filtered and the concentrations measured with non-suppressed anion exchange chromatography (Waters IC-Pak anion exchange column, waters 430 conductivity detector). The total sulfide concentrations (H2S + HS- + S**2-) were determined using the diamine complexation method (doi:10.4319/lo.1969.14.3.0454). Samples for dissolved inorganic carbon (DIC) and alkalinity measurements were preserved by adding 2 µl saturated mercury chloride (HgCl2) solution and stored headspace-free in gas-tight glass vials. DIC and alkalinity were measured using the flow injection method (detector VWR scientific model 1054) (doi:10.4319/lo.1992.37.5.1113). Dissolved sulfide was eliminated prior to the DIC measurement by adding 0.5 M molybdate solution (doi:10.4319/lo.1995.40.5.1011). Nutrient subsamples (10 - 15 ml) were stored at - 20 °C prior to concentration measurements with a Skalar Continuous-Flow Analyzer (doi:10.1002/9783527613984).

Pore water geochemistry of Håkon Mosby mud volcano sediments measured at station MSM16/2_847-1 (Z2-S2, aged flow surface)

Pore water and turnover rates were determined for surface sediment cores obtained in 2009 and 2010. The pore water was extracted with Rhizons (Rhizon CSS: length 5 cm, pore diameter 0.15 µm; Rhizosphere Research Products, Wageningen, Netherlands) in 1 cm-resolution and immediately fixed in 5% zinc acetate (ZnAc) solution for sulfate, and sulfide analyses. The samples were diluted, filtered and the concentrations measured with non-suppressed anion exchange chromatography (Waters IC-Pak anion exchange column, waters 430 conductivity detector). The total sulfide concentrations (H2S + HS- + S**2-) were determined using the diamine complexation method (doi:10.4319/lo.1969.14.3.0454). Samples for dissolved inorganic carbon (DIC) and alkalinity measurements were preserved by adding 2 µl saturated mercury chloride (HgCl2) solution and stored headspace-free in gas-tight glass vials. DIC and alkalinity were measured using the flow injection method (detector VWR scientific model 1054) (doi:10.4319/lo.1992.37.5.1113). Dissolved sulfide was eliminated prior to the DIC measurement by adding 0.5 M molybdate solution (doi:10.4319/lo.1995.40.5.1011). Nutrient subsamples (10 - 15 ml) were stored at - 20 °C prior to concentration measurements with a Skalar Continuous-Flow Analyzer (doi:10.1002/9783527613984).

Pore water geochemistry of arctic deep sea sediments measured at station MSM16/2_809-1 (REF, Reference)

Pore water and turnover rates were determined for surface sediment cores obtained in 2009 and 2010. The pore water was extracted with Rhizons (Rhizon CSS: length 5 cm, pore diameter 0.15 µm; Rhizosphere Research Products, Wageningen, Netherlands) in 1 cm-resolution and immediately fixed in 5% zinc acetate (ZnAc) solution for sulfate, and sulfide analyses. The samples were diluted, filtered and the concentrations measured with non-suppressed anion exchange chromatography (Waters IC-Pak anion exchange column, waters 430 conductivity detector). The total sulfide concentrations (H2S + HS- + S**2-) were determined using the diamine complexation method (doi:10.4319/lo.1969.14.3.0454). Samples for dissolved inorganic carbon (DIC) and alkalinity measurements were preserved by adding 2 µl saturated mercury chloride (HgCl2) solution and stored headspace-free in gas-tight glass vials. DIC and alkalinity were measured using the flow injection method (detector VWR scientific model 1054) (doi:10.4319/lo.1992.37.5.1113). Dissolved sulfide was eliminated prior to the DIC measurement by adding 0.5 M molybdate solution (doi:10.4319/lo.1995.40.5.1011). Nutrient subsamples (10 - 15 ml) were stored at - 20 °C prior to concentration measurements with a Skalar Continuous-Flow Analyzer (doi:10.1002/9783527613984).

Pore water geochemistry of Håkon Mosby mud volcano sediments measured at station MSM16/2_838-1 (Z1-S3, new flow surface)

Pore water and turnover rates were determined for surface sediment cores obtained in 2009 and 2010. The pore water was extracted with Rhizons (Rhizon CSS: length 5 cm, pore diameter 0.15 µm; Rhizosphere Research Products, Wageningen, Netherlands) in 1 cm-resolution and immediately fixed in 5% zinc acetate (ZnAc) solution for sulfate, and sulfide analyses. The samples were diluted, filtered and the concentrations measured with non-suppressed anion exchange chromatography (Waters IC-Pak anion exchange column, waters 430 conductivity detector). The total sulfide concentrations (H2S + HS- + S**2-) were determined using the diamine complexation method (doi:10.4319/lo.1969.14.3.0454). Samples for dissolved inorganic carbon (DIC) and alkalinity measurements were preserved by adding 2 µl saturated mercury chloride (HgCl2) solution and stored headspace-free in gas-tight glass vials. DIC and alkalinity were measured using the flow injection method (detector VWR scientific model 1054) (doi:10.4319/lo.1992.37.5.1113). Dissolved sulfide was eliminated prior to the DIC measurement by adding 0.5 M molybdate solution (doi:10.4319/lo.1995.40.5.1011). Nutrient subsamples (10 - 15 ml) were stored at - 20 °C prior to concentration measurements with a Skalar Continuous-Flow Analyzer (doi:10.1002/9783527613984).