Cytogenetic damage related to low levels of methyl mercury contamination in the Brazilian Amazon

The mercury rejected in the water system, from mining operations and lixiviation of soils after deforestation, is considered to be the main contributors to the contamination of the ecosystem in the Amazon Basin. The objectives of the present study were to examine cytogenetic functions in peripheral lymphocytes within a population living on the banks of the Tapajós River with respect to methylmercury (MeHg) contamination, using hair mercury as a biological indicator of exposure. Our investigation shows a clear relation between methylmercury contamination and cytogenetic damage in lymphocytes at levels well below 50 micrograms/gram, the level at which initial clinical signs and symptoms of mercury poisoning occur. The first apparent biological effect with increasing MeHg hair level was the impairment of lymphocyte proliferation measured as mitotic index (MI). The relation between mercury concentration in hair and MI suggests that this parameter, an indicator of changes in lymphocytes and their ability to respond to culture conditions, may be an early marker of cytotoxicity and genotoxicity in humans and should be taken into account in the preliminary evaluation of the risks to populations exposed in vivo. This is the first report showing clear cytotoxic effects of long-term exposure to MeHg. Although the results strongly suggest that, under the conditions examined here, MeHg is both a spindle poison and a clastogen, the biological significance of these observations are as yet unknown. A long-term follow-up of these subjects should be undertaken.

Mercury bioaccumulation in pike (Esox lucius) food chain from Anzali Lagoon, Iran

A number of wide-ranging monitoring studies have been performed in order to estimate the degree of mercury (Hg) contamination in freshwater ecosystems. Knowledge regarding contamination of different levels of the food chain is necessary for estimation of total pollutant input fluxes and subsequent partitioning among different phases in the aquatic system. The growing international concern about this environmental data is closely related to the strongly developing ecological risk assessment activities. In addition,freshwater monitoring outputs hold a key position in the estimation of the Hg dose consumed by the human population as it is highly dependent on fish consumption. So monitoring of Hg in the tissue of edible fish is extremely important because of contaminated fish has caused serious neurological damage to new born babies and adults. Mercury tends to accumulate in fish tissue, particularly, in the form of methyl mercury, which is about 10 times more toxic than inorganic mercury. The Anzali lagoon is one of the biggest wetland of Guilan province, which joins to the Caspian sea. Many Chemical and industrial factories plus agricultural runoffs and urban and rural sewages are major polluting sources of the Anzali wetland. Since many of those polluting sources drain their wastes directly or indirectly into the Anzali wetland and their sewages may be polluted with Hg, this study was conducted to find out the bioaccumulation of Hg bioaccumulation in pike (Esox lucius) food chain from Anzali lagoon, Iran. Sampling were carried out from July 2004 to July 2005, in addition 318 speciments of 9 fish species were collected. T-Hg was measured by LECO AMA 254 Advanced Mercury Analyzer (USA) according to ASTM standard No D-6722. Each sample was analyzed 3 times. Accuracy of T-Hg analysis was checked by running three samples of Standard Reference Materials; SRM 1633b, SRM 2711 & Sra 2709. Detection limit was 0.001 mg/kg in dry weight. The Accuracy degree of analyzor equipment with RSD<%0.05 (N=7) was between %95.5 and %105. In overal eigth fish species were distingushed in the gut content of 87 speciments of pike with age 1-5 year and maximum length 550mm. The max. and min. concentration of T-Hg in dorsal muscle of pjke was 0.2ppm in one year and 1.2ppm in five year class. The mean of T-Hg significantly increased with age and length increased (P<0.05).Mercury accumulation pattern in pike was as well as muscle > liver > spleen (P<0.05). THg content in female was higher than male(P<0.05). In contrast the mean of THg concentration in dorsal muscle of eigth fish species as prey was 0.282, 0.261, 0.328, 0.254, 0.256, 0.286, 0.322 and 0.241 ppm for Carassius auratus gibelio, Hemiculter leucisculus, Blicca bjoerkna transcaucasica, Chalcalburnus mossulensis, Rhodeus sericeus amarus, Gambusia holbrooki, Alburnus charusini hohenackeri & Scardinius Erythrophthalmus respectively.Liner regresion indicated that high degree of relationship between age of pike and Uptak/Intake ratio (R2=%99.12) and indicated that the mercury bioaccumulation in the pike dorsal muscle increased with age increased. BFA was >1 and and indicating the mercury biomagnification in the pike food chain. Trophy level of pike in the Anzali lagoon was estimated as well as 3.5 and 4 . It is generally agreed that Hg concentration in carnivorous fish are higher than in noncarnivorous species.

Effects of mercury intoxication on the response of horizontal cells of the retina of thraira fish (Hoplias malabaricus)

Methyl mercury (MeHg) is highly neurotoxic, affecting visual function in addition to other central nervous system functions. The effect of mercury intoxication on the amplitude of horizontal cell responses to light was studied in the retina of the fish Hoplias malabaricus. Intracellular responses were recorded from horizontal cells of fish previously intoxicated with MeHg by intraperitoneal injection (IP group) or by trophic exposure (T group). Only one retina per fish was used. The doses of MeHg chloride administered to the IP group were 0.01, 0.05, 0.1, 1.0, 2.0, and 6.0 mg/kg. The amplitudes of the horizontal cell responses were lower than control in individuals exposed to 0.01 (N = 4 retinas), 0.05 (N = 2 retinas) and 0.1 mg/kg (N = 1 retina), whereas no responses were recorded in the 1.0, 2.0, and 6.0 mg/kg groups. T group individuals were fed young specimens of Astyanax sp previously injected with MeHg corresponding to 0.75 (N = 1 retina), 0.075 (N = 8 retinas) or 0.0075 (N = 4 retinas) mg/kg fish body weight. After 14 doses, one every 5 days, the amplitude of the horizontal cell response was higher than control in individuals exposed to 0.075 and 0.0075 mg/kg, and lower in individuals exposed to 0.75 mg/kg. We conclude that intoxication with MeHg affects the electrophysiological response of the horizontal cells in the retina, either reducing or increasing its amplitude compared to control, and that these effects are related to the dose and/or to the mode of administration.

Fish Consumption and Prenatal Mercury Exposure

Methyl mercury levels in fish tissues have recently become a health issue. Does this toxin adversely affect humans to the point that fish consumption should be severely limited? Health effects of high levels of mercury exposure to human adults are fairly well known, however the effects of lower levels of exposure on human fetal development are less understood. Recent guidelines issued by the United States Food and Drug Agency and the Environmental Protection Agency recommended that pregnant women should refrain from consuming some types of fish, and limit consumption to certain levels, but results of health studies used to develop recommendations were inconclusive when low levels of in-utero methyl mercury exposure were compared. Other studies demonstrated health benefits of fish consumption to developing fetuses. These health benefits may be an over-riding factor, and my interpretation of various studies concludes that restricted consumption of some fish species may be premature at this time due to a need to balance potential health benefits against mercury toxicity hazard.

Avaliação da exposição ao metil-mercúrio na população ribeirinha da baixada cuiabana, no Pantanal Mato-Grossense, Brasil

Os efeitos tóxicos do metil-mercúrio (MeHg) em seres humanos vêm sendo amplamente discutidos, a partir de episódios de contaminação devida a ingestão frequente de peixe contaminado, em Minamata, no Japão, ou ao consumo de pão preparado com cereais tratados com fungicidas a base de metil-mercúrio, no Iraque, indicando que o metil mercúrio pode afetar o sistema nervoso central. A população ribeirinha na região do Pantanal, Brasil, é dependente do peixe, como principal fonte de alimentos e proteínas. Naquela região, o resultado de décadas de mineração de ouro, em pequena escala, foi a contaminação de muitos sistemas aquáticos com mercúrio. Consequentemente, muitas espécies de peixes possuem níveis de MeHg relativamente altos. O objetivo deste trabalho foi avaliar o nível de exposição ao MeHg entre a população ribeirinha do Pantanal. No primeiro artigo, apresentam-se os resultados da validação do método recordatório de 24 horas auto-referido, utilizando-se, como padrão-ouro, o método da pesagem, tendo-se verificado que há aproximadamente um erro de 30% ao estimar a quantidade de peixe consumido por aquela população. Um erro deste pode ser importante em se tratando do consumo de peixes carnívoros, os quais apresentam altos teores de mercúrio. No segundo artigo, ao analisar a associação entre o status neurocognitivo e a concentração de mercúrio no cabelo da população acima, os resultados indicaram que adultos expostos ao metil-mercúrio, através do consumo de peixe, podem ter déficits importantes nas medidas do desempenho neurocomportamental, sem alterações detectáveis no humor ou afetividade. Os efeitos mais importantes do mercúrio, entre os indivíduos analisados, foram detectados nos testes de velocidade e destreza da coordenação motora fina, na inibição da resposta na busca visual, e em tarefas de atenção. No terceiro artigo, considerando-se as associações observadas na concentração de mercúrio no cabelo com alguns desfechos dos testes neuro-psicológicos aplicados, calculou-se a dose-marcadora (BMD) e o limite inferior do intervalo de confiança do BMD (BMDL). Os resultados da dose marcadora e da dose de referência provisória são concordantes com os resultados estimados pelas agências de saúde internacionais.

Determination of Hg(II) in waters by on-line preconcentration using Cyanex 923 as a sorbent - Cold vapor atomic absorption spectrometry

Using a solid phase extraction mini-column home-made from a neutral extractant Cyanex 923, inorganic Hg could be on-line preconcentrated and simultaneously separated from methyl mercury. The preconcentrated Hg (11) was then eluted with 10% HNO3 and subsequently reduced by NaBH4 to form Hg vapor before determination by cold vapor atomic absorption spectrometry (CVAAS). Optimal conditions for and interferences on the Hg preconcentration and measurement were at 1% HCl, for a 25 mL sample uptake volume and a 10 mL min(-1) sample loading rate. The detection limit was 0.2 ng L-1 and much lower than that of conventional method (around 15.8 ng L-1). The relative standard deviation (RSD) is 1.8% for measurements of 40 ng L-1 of Hg and the linear working curve is from 20 to 2000 ng L-1 (with a correlation coefficient of 0.9996). The method was applied in determination of inorganic Hg in city lake and deep well water (from Changchun, Jilin, China), and recovery test results for both samples were satisfactory.

Relation entre les espèces de mercure, le sélénium et les thiols dans les eaux de surface du parc national du Mont-Tremblant (Laurentides, Québec)

Les thiols et le sélénium peuvent jouer un rôle important dans la méthylation du mercure des environnements aquatiques. Pour démontrer la présence des thiols et du sélénium et leur relation avec le mercure dans certains écosystèmes d'eau douce québécois, une campagne d’échantillonnage fut réalisée durant l’été 2010, dans le parc national du Mont-Tremblant (Laurentides, Québec). Il existe une corrélation significative entre le sélénium et le mercure total dans l’eau des lacs du parc. Cependant, les concentrations de sélénium sont très faibles dans les lacs, les étangs de castor et les ruisseaux. Par ailleurs, les lacs du parc national du Mont-Tremblant ont des concentrations relativement élevées de méthylmercure avec une moyenne de 0,33 ng L-1 et des maximums allant jusqu’à 3,29 ng L-1. Les étangs de castor peuvent aussi être considérés comme des lieux de contamination au méthylmercure, avec une concentration moyenne de 0,95 ng L-1. Toutefois, la présence d’une colonie de castors sur le bassin versant d’un lac ne semble pas influencer les concentrations de mercure que l’on y retrouve. Deux thiols sont détectables dans l’eau de surface des Laurentides, soit le glutathion et l’acide thioglycolique. La concentration de ce dernier thiol est corrélée significativement avec celle du mercure total et du méthylmercure. Les thiols peuvent jouer un rôle important dans les processus de méthylation en favorisant le transport du mercure inorganique à l’intérieur des bactéries sulfato-réductrices. Afin de mieux comprendre l’action antagoniste entre le sélénium et le mercure, des études devraient être réalisées au niveau des tissus des organismes vivants dans ces zones pauvres en sélénium.

Use of Cathorops spixii as bioindicator of pollution of trace metals in the Santos Bay, Brazil

In the present study Cathorops spixii, was evaluated as a bioindicator fish for trace metal pollution. Concentrations of cobalt (Co), iron (Fe), selenium (Se) and zinc (Zn) were determined by Instrumental Neutron Activation Analysis in liver. Mercury (Hg) and methyl-mercury (MeHg) were analyzed by Cold Vapor Atomic Absorption Spectrometry in muscles and livers. High concentrations of Co, Fe, Se and Zn were observed in C. spixii from Santos Bay in comparison to fish collected in a non-polluted site in the same Brazilian coast. These trace metal concentrations were out of the permissible levels for human consumption. Although, Hg and MeHg levels were low, the C. spixii could still be used as an effective bioindicator to observe trace metal behaviors in the environment in function of the bioaccumulation process observed mainly by other analyzed trace metals. Thus, the use of this species is strongly recommended to monitor the effects and behavior of trace metal pollution in aquatic ecosystems in Brazil due to its bioaccumulation function.

In vivo and in vitro antioxidant activity and hepatoprotective properties of polyphenols from Halimeda opuntia (Linnaeus) Lamouroux

Antioxidant activity and hepatoprotective properties of the aqueous extract and tetrahydrofuran-extracted phenolic fractions of Halimeda opuntia (Linnaeus) Lamouroux were investigated in rats with chemically induced liver injury. Total polyphenols were determined by using the Folin-Ciocalteau reagent. Liver damage was induced by CCl4 and assessed by a histological technique. Reverse transcription/polymerase chain reaction (RT/PCR) analysis showed increased superoxide dismutase (SOD) and catalase (CAT) gene expression and activities in the group treated with free phenolic acid (FPA) fractions of H. opuntia, suggesting inducing effects on both enzymes. In addition, rats treated with FPA fractions displayed lower liver thiobarbituric acid reactive substance (TBARS) levels than those observed for rats in the CCl4-treated group. These data suggest that the phenolic fractions from H. opuntia may protect the liver against oxidative stress-inducing effects of chemicals by modulating its antioxidant enzymes and oxidative status.

ANALYSIS OF IN VITRO AND IN VIVO ANTIOXIDANT PROPERTIES OF HYDROPHILIC FRACTIONS FROM THE SEAWEED HALIMEDA MONILE L.

In this work, in vitro and in vivo antioxidant properties of the marine algae Halimeda monile were assessed and the levels of some of its compounds likely to be responsible for such properties were determined. The estimated contents of total polyphenols, chlorophylls a and b and carotenoids were 179.5, 356.3, 452.8 and 42.2 mu g/g dry weight seaweed, respectively. The presence of terpenoids and flavonoids was also observed. The antioxidant activity of two polar fractions from H. monile (lyophilized aqueous extract and free phenolic acid fraction) was evaluated using three antioxidant assays: ferric reducing antioxidant power, 1,1-diphenyl-2-picrylhydrazyl and lipid peroxidation. Treatment of CCl4-induced liver damage in rats with extracts resulted in lower serum thiobarbituric acid-reactive substances levels and higher hepatic glutathione concentrations compared to those observed in the CCl4-treated group. Also, a significant increase in catalase activity was detected after treatment with the extracts. These results suggest that the seaweed H. monile could be a potential source for natural antioxidants.

Oral exposure to methylmercury modifies the prostatic microenvironment in adult rats

Methylmercury (MeHg) is an environmental pollutant that is highly toxic to the central nervous system. As its effects on male reproductive system are poorly understood, this study was carried out to analyse the effects of MeHg on the rat prostate. To evaluate the MeHg toxicity on ventral prostate, three groups of adult male Wistar rats received oral doses of 0.5, 1.0 and 3.0mg/kg MeHg, respectively, on a daily basis for 14days. A fourth group was used as a control. The prostate weight was decreased in rats treated orally with 0.5mg/kg MeHg compared to controls. Also, Hg concentration increased significantly in the prostate after treatments. There were reductions in serum testosterone levels and androgen receptor immunoreactivity in animals receiving 3.0mgMeHg/kg. The stereological data showed changes in the prostatic epithelial, stromal and luminal compartments which varied according to the different doses. Histopathological alterations, such as chronic inflammation, stratified epithelial hyperplasia and epithelial inflammatory reactive atypia, were observed in the 0.5mg/kg MeHg-treated group. Epithelial atrophy was observed in the 3.0mg/kg MeHg-treated group. In conclusion, the MeHg affects prostatic homoeostasis resulting in histopathological changes that may be relevant in the pathogenesis of prostatic disease.

Morphological evidence of neurotoxicity in retina after methylmercury exposure

The visual system is particularly sensitive to methylmercury (MeHg) exposure and, therefore, provides a useful model for investigating the fundamental mechanisms that direct toxic effects. During a period of 70 days, adult of a freshwater fish species Hoplias malabaricus were fed with fish prey previously labeled with two different doses of methylmercury (0.075 and 0.75 mu g g(-1)) to determine the mercury distribution and morphological changes in the retina. Mercury deposits were found in the photoreceptor layer, in the inner plexiform layer and in the outer plexiform layer, demonstrating a dose-dependent bioaccumulation. The ultrastructure analysis of retina revealed a cellular deterioration in the photoreceptor layer, morphological changes in the inner and outer segments of rods, structural changes in the plasma membrane of rods and double cones, changes in the process of removal of membranous discs and a structural discontinuity. These results lead to the conclusion that methylmercury is able to cross the blood-retina barrier, accumulate in the cells and layers of retina and induce changes in photoreceptors of H. malabaricus even under subchronic exposure. (c) 2012 Elsevier Inc. All rights reserved.

Massenspektrometrische Isotopenverdünnungsanalyse (MSIVA) als Validierungsmethode für Bestimmungen von Methylquecksilber mittels GC/ICP-MS

Im Rahmen dieser Arbeit ist es gelungen, ein GC/ICP-MSIVA-Verfahren zur zuverlässigen und richtigen Bestimmung von Methylquecksilbergehalten (MeHg+) in aquatischen Systemen zu entwickeln. Unter Verwendung eines hergestellten Me201Hg+-Indikators konnte bei der Überprüfung der weltweit gängigen Probenaufbereitung mittels NaBEt4 zur Überführung von MeHg+ in MeEtHg gezeigt werden, daß während dieser Probenaufbereitung in Gegenwart von Halogeniden eine MeHg+-Speziesumwandlung zu Hg0 stattfinden kann. Es konnte mit Hilfe von Modellösungen erstmalig eindeutig nachgewiesen werden, daß die MeHg+-Speziesumwandlungen ihre Ursache nur im Zusammenwirken von vorliegenden Halogeniden mit dem verwendeten Derivatisierungsreagenz NaBEt4 haben. Ebenso wie in Modellösungen wurden auch MeHg+-Speziesumwandlungen in Realproben bei der Derivatisierung mit NaBEt4 überprüft. Die Proben, die bei der Derivatisierung mit NaBEt4 eine MeHg+-Umwandlung zeigten, wurden auch mit dem weltweit bisher wenig eingesetzten Reagenz NaBPr4 derivatisiert. Hier konnte bei keiner der Untersuchungen eine MeHg+-Umwandlung festgestellt werden. Des weiteren wurden MeHg+-Gehalte in Süßwasserproben unter Verwendung von NaBEt4 und NaBPr4 bestimmt. Die Ergebnisse hinsichtlich beider Derivatisierungsreagenzien stimmten sehr gut überein. Diese Resultate zeigten eindrucksvoll, daß Substanzverluste durch die Speziesumwandlung bei MeHg+-Bestimmungen mit MSIVA keine Rolle spielen, da die Umwandlung erst nach der Isotopenvermischung einsetzte. Mit dem entwickelten GC/ICP-MSIVA-Verfahrens wurde der MeHg+-Gehalt im Referenzmaterial CRM 463 analysiert, der sehr gut mit dem zertifizierten Wert übereinstimmte.

Konzeptionelle Entwicklung von Mikrokosmos-Experimenten zur Untersuchung der speziesspezifischen Transformationsprozesse des Quecksilbers mittels Isotopenverhältnisbestimmungen

Zur Untersuchung der speziesspezifischen Transformationsprozesse des Quecksilbers in der Umwelt wurden erstmalig Mikrokosmosexperimente unter Verwendung von isotopenangereicherten Verbindungen durchgeführt. Es wurden naturrelevante Bedingungen simuliert, um eine spätere Übertragbarkeit der Ergebnisse auf den biogeochemischen Kreislauf des Quecksilbers zu gewährleisten. Die aufgebauten Mikrokosmen bestanden aus Boden/Pflanzen/Luft-Kompartimenten. Der Boden der Mikrokosmen wurde mit isotopenangereicherten Quecksilberspezies dotiert. Durch die Verwendung von isotopenangereicherten Verbindungen, verbunden mit gleichzeitiger ICP/MS-Detektion, konnten auftretende Transformationsprozesse beobachtet werden. Die Messung der Quecksilberspezies erfolgte mittels GC-ICP/MS nach vorheriger Derivatisierung mit Natriumtetraethylborat und Anreicherung per 'purge and trap'.Die Massenspuren der einzelnen Quecksilberisotope wurden für alle Quecksilberspezies gemessen und daraus dann für jede Spezies die Isotopenverhältnisse gebildet. Bei einer Änderung des Isotopenverhältnisses kann von einer Speziestransformation ausgegangen werden.Aus den Mikrokosmosexperimenten, denen Methylquecksilber als angereicherte Isotopenverbindung zugegeben wurde, konnte gefolgert werden, dass Methylquecksilber im Boden zunächst zu anorganischem Quecksilber demethyliert wurde. Im Anschluss daran erfolgte eine Reduktion zu elementarem Quecksilber. Dieses gebildete elementare Quecksilber verflüchtigte sich nahezu vollständig (ca. 90-100%) vom Boden in die Atmosphäre.Bei der Zugabe von anorganischem Quecksilber als angereicherte Isotopenverbindung in den Boden wurde vorwiegend eine Reduktion zu elementarem Quecksilber beobachtet, das dann in die Atmosphäre emittiert. Es konnte aber auch eine geringe Methylierung (ca. 5%) zu Methylquecksilber beobachtet werden. Daraus kann gefolgert werden, dass die methylierten Quecksilberverbindungen eine wesentliche Rolle im natürlichen Kreislauf des Quecksilbers spielen.Parallel zu den Mikrokosmosexperimenten wurden Feldversuche in einem flussnahen Feuchtgebiet, aus dem auch der Boden für die Mikrokosmosexperimente entnommen worden war, durchgeführt. In den Feldversuchen wurden Quecksilberkonzentrationen und der Quecksilberfluss zwischen dem Boden und der Atmosphäre mit Hilfe von Flusskammerexperimenten bestimmt. Es konnten mit Hilfe von Mikrokosmen, die natürliche Verhältnisse simulieren sollten, und mit Hilfe verschieden angereicherter Isotopenspikes erstmals Speziestransformationen des Quecksilbers direkt beobachtet werden. Die GC-ICP/MS-Methode ermöglichte eine eindeutige Identifikation von Edukt und Produkt der jeweiligen Umwandlung. Allerdings wurde die Untersuchung eingeschränkt durch die irreversiblen biologischen Veränderungen in den eingesetzten Mikrokosmen nach über einer Woche und durch die Notwendigkeit, vergleichsweise hohe Konzentrationen der Spikes einzusetzen, um eine statistisch signifikante Auswertung der Veränderung der Isotopenverhältnisse zu erreichen. Somit sind Mikrokosmosexperimente nur eingeschränkt für Untersuchungen des Quecksilberverhaltens geeignet.Im Rahmen dieser Arbeit ist es aber gelungen, die Mikrokosmenexperimente unter Verwendung von isotopenangereicherten Verbindungen und der GC-ICP/MS-Methode als leistungsstarkes Verfahren zur Beobachtung von Speziestransformationsprozessen des Quecksilbers zu etablieren.