Short exposure to ethyl and methylmercury prompts similar toxic responses in Drosophila melanogaster.

Methylmercury (MeHg) and ethylmercury (EtHg) are important mercury organic forms in terms of human poisoning. Since the comparative effects of compounds are mainly in vitro, this study was designed to investigate the toxicities induced by MeHg and EtHg in an in vivo study using adult Drosophila melanogaster (D. melanogaster). Firstly, we performed a survival curve, where the flies were fed on a medium containing MeHg and EtHg at concentrations ranging from 2.5 to 200 µM, until the end of their lifespan. After that, the concentrations 25 and 200 µM of MeHg and EtHg were chosen to be tested in a short exposure for 5 days. The analysis of survival by Kaplan-Meier plot revealed that all concentrations of MeHg and EtHg reduced significantly the lifespan of the flies. Short exposure to both concentrations of MeHg and EtHg impaired the ability of flies in the climbing assay and induced lipid peroxidation. Only the flies exposed to the highest concentration had viability loss, thiol depletion, and increased reactive species (RS) and Hg levels in the whole body. Our findings indicate that MeHg and EtHg exhibit similar toxic effects in vivo, and that oxidative stress is a phenomenon behind the toxicity of both mercurials. The data obtained also reinforce the use of D. melanogaster as a useful organism for basic toxicological research.

Heterogeneity of Multimedia Exposures to Neurotoxic Elements (Al, As, Cd, Pb, Mn, and Hg) in Breastfed Infants from Porto Velho, Brazil.

Infant exposure to neurotoxic elements is a public health issue that needs monitoring with regard to breast milk composition. We studied six neurotoxic elements in breast milk samples at different stages of lactation in mothers from Porto Velho, Brazil. We used a flow-injection mercury system (FIMS) to determine total Hg concentrations and an inductively coupled plasma optical emission spectrometer (ICP-OES) to determine the concentrations of Al, As, Cd, Pb, and Mn in 106 donors of a human milk bank. Association rules analyses were applied to determine the pattern of binary and ternary mixtures of the measured exposants. The metal concentration was mostly below the limit of detection (LOD) for Cd (99%), Pb (84%), and Hg (72%), and it was above the LOD for As (53%), Mn (60%), and Al (82%), respectively. Median concentrations (dry weight) of Al, As, Hg, Mn, and Pb were 1.81 µg/g, 13.8 ng/g, 7.1 ng/g, 51.1 ng/g, and 0.43 µg/g, respectively. Al is singly the most frequent element to which infants are exposed. Occurring binary combination (> LOD) was 56% for Al-Mn, 41% for Al-As, 22% for Al-Hg, and 13% for Al-Pb. In 100% of neonates, exposure to Al-ethylmercury (EtHg) occurred through immunization with thimerosal-containing vaccines (TCV). Association rules analysis revealed that Al was present in all of the multilevel combinations and hierarchical levels and that it showed a strong link with other neurotoxic elements (especially with Mn, As, and Hg). (a) Nursing infants are exposed to combinations of neurotoxicants by different routes, dosages, and at different stages of development; (b) In breastfed infants, the binary exposures to Al and total Hg can occur through breast milk and additionally through TCV (EtHg and Al);

The toxicology of mercury: Current research and emerging trends.

Mercury (Hg) is a persistent bio-accumulative toxic metal with unique physicochemical properties of public health concern since their natural and anthropogenic diffusions still induce high risk to human and environmental health. The goal of this review was to analyze scientific literature evaluating the role of global concerns over Hg exposure due to human exposure to ingestion of contaminated seafood (methyl-Hg) as well as elemental Hg levels of dental amalgam fillings (metallic Hg), vaccines (ethyl-Hg) and contaminated water and air (Hg chloride). Mercury has been recognized as a neurotoxicant as well as immunotoxic and designated by the World Health Organization as one of the ten most dangerous chemicals to public health. It has been shown that the half-life of inorganic Hg in human brains is several years to several decades. Mercury occurs in the environment under different chemical forms as elemental Hg (metallic), inorganic and organic Hg. Despite the raising understanding of the Hg toxicokinetics, there is still fully justified to further explore the emerging theories about its bioavailability and adverse effects in humans. In this review, we describe current research and emerging trends in Hg toxicity with the purpose of providing up-to-date information for a better understanding of the kinetics of this metal, presenting comprehensive knowledge on published data analyzing its metabolism, interaction with other metals, distribution, internal doses and targets, and reservoir organs.

Alkyl Mercury-Induced Toxicity: Multiple Mechanisms of Action.

There are a number of mechanisms by which alkylmercury compounds cause toxic action in the body. Collectively, published studies reveal that there are some similarities between the mechanisms of the toxic action of the mono-alkyl mercury compounds methylmercury (MeHg) and ethylmercury (EtHg). This paper represents a summary of some of the studies regarding these mechanisms of action in order to facilitate the understanding of the many varied effects of alkylmercurials in the human body. The similarities in mechanisms of toxicity for MeHg and EtHg are presented and compared. The difference in manifested toxicity of MeHg and EtHg are likely the result of the differences in exposure, metabolism, and elimination from the body, rather than differences in mechanisms of action between the two.

From the Cover: Ethylmercury-Induced Oxidative and Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death: Involvement of Autophagosome-Lysosome Fusion Arrest.

Ethylmercury (EtHg) is derived from the degradation of thimerosal, the most widely used organomercury compound. In this study, EtHg-induced toxicity and autophagy in the mouse kidney was observed and then the mechanism of toxicity was explored in vitro in HK-2 cells. Low doses of EtHg induced autophagy without causing any histopathological changes in mouse kidneys. However, mice treated with high doses of EtHg exhibited severe focal tubular cell necrosis of the proximal tubules with autophagy. EtHg dose-dependently increased the production of reactive oxygen species, reduced the mitochondrial membrane potential, activated the unfolded protein response, and increased cytosolic Ca2+ levels in HK-2 cells. Cell death induced by EtHg exposure was caused by autophagy and necrosis. N-acetyl cysteine and 4-phenylbutyric acid attenuated EtHg-induced stress and ameliorated the autophagic response in HK-2 cells. Furthermore, EtHg blocked autophagosome fusion with lysosomes, which was demonstrated via treatment with wortmannin and chloroquine. Low doses of EtHg and rapamycin, which resulted in minimal cytotoxicity, increased the levels of the autophagic SNARE complex STX17 (syntaxin 17)-VAMP8-SNAP29 without altering mRNA levels, but high dose of EtHg was cytotoxic. Inhibition of autophagic flux by chloroquin increased autophagosome formation and necrotic cell death in HK-2 cells. Collectively, our results show that EtHg induces autophagy via oxidative and ER stress and blockade of autophagic flux. Autophagy might play a dual role in EtHg-induced renal toxicity, being both protective following treatment with low doses of EtHg and detrimental following treatment with high doses.

Ethylmercury and Hg2+ induce the formation of neutrophil extracellular traps (NETs) by human neutrophil granulocytes.

Humans are exposed to different mercurial compounds from various sources, most frequently from dental fillings, preservatives in vaccines, or consumption of fish. Among other toxic effects, these substances interact with the immune system. In high doses, mercurials are immunosuppressive. However, lower doses of some mercurials stimulate the immune system, inducing different forms of autoimmunity, autoantibodies, and glomerulonephritis in rodents. Furthermore, some studies suggest a connection between mercury exposure and the occurrence of autoantibodies against nuclear components and granulocyte cytoplasmic proteins in humans. Still, the underlying mechanisms need to be clarified. The present study investigates the formation of neutrophil extracellular traps (NETs) in response to thimerosal and its metabolites ethyl mercury (EtHg), thiosalicylic acid, and mercuric ions (Hg(2+)). Only EtHg and Hg(2+) triggered NETosis. It was independent of PKC, ERK1/2, p38, and zinc signals and not affected by the NADPH oxidase inhibitor DPI. Instead, EtHg and Hg(2+) triggered NADPH oxidase-independent production of ROS, which are likely to be involved in mercurial-induced NET formation. This finding might help understanding the autoimmune potential of mercurial compounds. Some diseases, to which a connection with mercurials has been shown, such as Wegener's granulomatosis and systemic lupus erythematosus, are characterized by high prevalence of autoantibodies against neutrophil-specific auto-antigens. Externalization in the form of NETs may be a source for exposure to these self-antigens. In genetically susceptible individuals, this could be one step in the series of events leading to autoimmunity.

Toxicological effects of thiomersal and ethylmercury: Inhibition of the thioredoxin system and NADP(+)-dependent dehydrogenases of the pentose phosphate pathway.

Mercury (Hg) is a strong toxicant affecting mainly the central nervous, renal, cardiovascular and immune systems. Thiomersal (TM) is still in use in medical practice as a topical antiseptic and as a preservative in multiple dose vaccines, routinely given to young children in some developing countries, while other forms of mercury such as methylmercury represent an environmental and food hazard. The aim of the present study was to determine the effects of thiomersal (TM) and its breakdown product ethylmercury (EtHg) on the thioredoxin system and NADP(+)-dependent dehydrogenases of the pentose phosphate pathway. Results show that TM and EtHg inhibited the thioredoxin system enzymes in purified suspensions, being EtHg comparable to methylmercury (MeHg). Also, treatment of neuroblastoma and liver cells with TM or EtHg decreased cell viability (GI50: 1.5 to 20µM) and caused a significant (p<0.05) decrease in the overall activities of thioredoxin (Trx) and thioredoxin reductase (TrxR) in a concentration- and time-dependent manner in cell lysates. Compared to control, the activities of Trx and TrxR in neuroblastoma cells after EtHg incubation were reduced up to 60% and 80% respectively, whereas in hepatoma cells the reduction was almost 100%. In addition, the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also significantly inhibited by all mercurials, with inhibition intensity of Hg(2+)>MeHg≈EtHg>TM (p<0.05). Cell incubation with sodium selenite alleviated the inhibitory effects on TrxR and glucose-6-phosphate dehydrogenase. Thus, the molecular mechanism of toxicity of TM and especially of its metabolite EtHg encompasses the blockage of the electrons from NADPH via the thioredoxin system.

Are neuropathological conditions relevant to ethylmercury exposure?

Mercury and mercurial compounds are among the environmentally ubiquitous substances most toxic to both wildlife and humans. Once released into the environment from both natural and anthropogenic sources, mercury exists mainly as three different molecular species: elemental, inorganic, and organic. Potential health risks have been reported from exposure to all forms; however, of particular concern for human exposure relate to the potent neurotoxic effects of methylmercury (MeHg), especially for the developing nervous system. The general population is primarily exposed to MeHg by seafood consumption. In addition, some pharmaceuticals, including vaccines, have been, and some continue to be, a ubiquitous source of exposure to mercurials. A significant controversy has been whether the vaccine preservative ethylmercury thiosalicylate, commonly known as thimerosal, could cause the development of autism. In this review, we have discussed the hypothesis that exposure to thimerosal during childhood may be a primary cause of autism. The conclusion is that there are no reliable data indicating that administration of vaccines containing thimerosal is a primary cause of autism. However, one cannot rule out the possibility that the individual gene profile and/or gene-environment interactions may play a role in modulating the response to acquired risk by modifying the individual susceptibility.

Kawasaki's disease, acrodynia, and mercury.

A superantigen or autoimmunity has been hypothesized to be the main cause of the Kawasaki's Disease but the etiology is unknown. Medical literature, epidemiological findings, and some case reports have suggested that mercury may play a pathogenic role. Several patients with Kawasaki's Disease have presented with elevated urine mercury levels compared to matched controls. Most symptoms and diagnostic criteria which are seen in children with acrodynia, known to be caused by mercury, are similar to those seen in Kawasaki's Disease. Genetic depletion of glutathione S-transferase , a susceptibility marker for Kawasaki's Disease, is known to be also a risk factor for acrodynia and may also increase susceptibility to mercury . Coinciding with the largest increase (1985-1990) of thimerosal (49.6% ethyl mercury) in vaccines, routinely given to infants in the U.S. by 6 months of age (from 75microg to 187.5microg), the rates of Kawasaki's Disease increased ten times, and, later (1985-1997), by 20 times. Since 1990 88 cases of patients developing Kawasaki's Disease some days after vaccination have been reported to the Centers of Disease Control (CDC) including 19% manifesting symptoms the same day. The presented pathogenetic model may lead to new preventive- and therapeutic strategies for Kawasaki's disease.

When science is not enough - a risk/benefit profile of thiomersal-containing vaccines.

Without a preservative, such as thiomersal (known as thimerosal in the US), multi-dose liquid presentations of vaccine are vulnerable to bacteriological contamination that can result in death or serious illness of the recipient. Concerns about levels of mercury exposure from thiomersal-containing vaccines were first raised in the US during 1999 in the context of Hepatitis B vaccine for newborns. Since then, a large body of evidence from animal and epidemiological studies has accumulated on the safety of thiomersal. Ironically, these data have become largely irrelevant in wealthy countries, where mono-dose, thiomersal-free vaccines have been introduced as a precautionary measure in almost all childhood vaccines, in part related to residual public scepticism. In poor countries, multi-dose vials remain important for vaccine delivery. There is a real danger that this controversy may result in the loss to the world of thiomersal as a preservative, simply from popular pressure. In reality, it would be impossible to cease overnight using thiomersal and maintain the supply of vital vaccines. This paper reviews and summarises the data available from published studies on mercury toxicity, and thiomersal in vaccines in particular, that overwhelmingly indicate continued use of thiomersal is safe in those countries where it is most needed.

The evidence for the safety of thiomersal in newborn and infant vaccines.

While a number of studies remain to be completed, evidence is mounting that there is no demonstrable risk for infants immunized with vaccines containing thiomersal. Epidemiological studies in the US have shown no developmental or other central nervous system abnormalities resulting from exposure to vaccines containing thiomersal. During the initial evaluation of thiomersal in vaccines during 1999, the toxicological profile of ethyl mercury was unknown and presumed to be the same as that of methyl mercury. Enough evidence has accumulated since then to indicate the profiles of the two compounds are different in crucial aspects. To date, one study has measured blood levels of total mercury in vaccinated infants and reports only a brief low-level exposure with rapid excretion of mercury. It is not yet known for sure how much (if any) vaccine-derived ethyl mercury in the blood crosses the blood-brain barrier. For the most part, the use of thiomersal as a vaccine preservative has been convincingly shown to be safe. The scientific evidence is not yet sufficiently strong to provide the same level of assurance for thiomersal-containing vaccines for use in pregnant women or the premature or low birth weight infant. There is an increased sensitivity of the fetal brain to mercury whether it is ethyl or methyl mercury. While there is no evidence to support the contention, it is at least theoretically possible that very low birth weight premature infants may be at increased risk from thiomersal-containing vaccines. Until such time as the scientific evidence is to hand, thiomersal-free presentations of hepatitis B are to be preferred for the birth dose. Given the same levels of exposure, adults are at much lower levels of risk because of increased body mass. It is not possible to prove that thiomersal is completely safe-epidemiology can only quantify a risk, not prove its absence.

[The effect of the duration of methoxyethylmercury acetate exposure on the lymphocytes in the peripheral blood of carp (Cyprinus carpio L.)]. / Der Einfluss der Dauer der Methoxyethylquecksilberazetatexposition auf die Lymphozyten im peripheren Blut des Karpfens (Cyprinus carpio L.).

The purpose of this work was to study possible hematological changes in carp (Cyprinus carpio L.) which might be induced by the experimental poisoning with metoxyethylmercury acetate which is used as fungicide. The experimental group of fishes was exposed to the named substance which was mixed with the feed during three weeks. The total lymphocytes number decreased in experimental group already at 7th day (p < 0.01), and remained almost at the same level, but considerably lower than the experimental group, until the end of the experiment. The number of small lymphocytes in the experimental group decreased also at the 7th day of the experiment (p < 0.01), and the decreasing tendency continued up to the end of the experiment. The number of large lymphocytes increased at the day 7th of the experiment (p < 0.01), and the increasing tendency continued until the end of the experiment. The population of large lymphocytes showed the cytopathological changes in the form of cytoplasmic vacuoles and cytoplasmic shoots. The frequency of these changes increased through the experiments.

Potentiating effects of organomercuries on clastogen-induced chromosome aberrations in cultured Chinese hamster cells.

Mercury compounds are among the most serious environmental pollutants. In this communication, the potentiating effects of organic and inorganic mercuries on clastogen-induced chromosome aberrations were studied in Chinese hamster CHO K1 cells. Post-treatment with monoalkylated mercuries--methyl mercuric chloride (MeHgCl) and ethyl mercuric chloride (EtHgCl)--increased the number of breakage- and exchange-type aberrations induced by 4-nitroquinoline 1-oxide (4NQO) and methyl methanesulfonate. With the DNA crosslinking agents mitomycin C (MMC) and cisplatin, MeHgCl enhanced both types of aberrations while EtHgCl enhanced breakage-type aberrations only. Since these monoalkylated mercuries did not show clastogenic effects by themselves under the present experimental conditions, the increases in chromosome aberrations were not additive. Dialkylated mercuries (dimethyl mercury and diethyl mercury) and inorganic mercuries (HgCl and HgCl2) did not show any potentiating effects. When MMC- or 4NQO-treated cells were post-treated with MeHgCl during the G1 phase, both breakage- and exchange-type aberrations were enhanced. Treatment with EtHgCl during the G1 phase also enhanced both types of aberrations induced by 4NQO. With MMC, however, G1 treatment with EtHgCl did not show any potentiating effect. MeHgCl and EtHgCl treatments during the G2 phase enhanced breakage-type aberrations only. Based on these results, the following possible mechanisms for potentiation of clastogenicity by monoalkylated mercuries were suggested; (1) they interfere with repair of base lesions induced by 4NQO and MMS during the pre-replicational stage, thereby increasing unrepaired DNA lesions which convert into DNA double-strand breaks in S phase, (2) MeHgCl (but not EtHgCl) also inhibits repair of crosslinking lesions during the pre-replicational stage, and (3) their G2 effects enhance breakage-type aberrations only.

Effect of organic and inorganic mercuric salts on Na+K+ATPase in different cerebral fractions in control and intrauterine growth-retarded rats: alterations induced by serotonin.

An intrauterine growth-retarded (IUGR) model based on restriction of blood supply to the rat fetus at the 17th day of pregnancy was studied. We investigated in vitro the effects of thimerosal and mercuric chloride on Na+K+ATPase activity in total brain homogenate, synaptosomes and myelin at weaning. In addition, we evaluated the reversal effect of serotonin on mercury-inhibited Na+K+ATPase activity. The toxicity, in terms of inhibition of Na+K+ATPase activity was greater with mercuric chloride than with thimerosal. Synaptosomes and principally myelin were more sensitive to the metal salts than total homogenate. Serotonin stimulated the Na+K+ATPase activity in total brain homogenate and synaptosomes but inhibited the enzyme in the myelin fraction. This effect was more marked in the IUGR group than in the control group. Serotonin (1 mM) added to total homogenate pretreated with the mercury salts produced variable reversal effects. In the synaptosomal fraction reverse effect was noted with serotonin. In myelin fraction, added serotonin increased inhibition caused by thimerosal.

Toxic effects of ophthalmic preservatives on cultured rabbit corneal epithelium.

We investigated the effects of the ophthalmic preservatives thimerosal and sorbic acid on the proliferation and survival of rabbit corneal epithelial cells in tissue culture. Normally, explants of corneal epithelium grow vigorously during the first 7 days in culture. With 0.004% thimerosal present in the culture medium, the normal proliferation of corneal cells is suppressed completely. When 0.1% sorbic acid is present, proliferation is delayed and the lifespan of the corneal cells is reduced. After a 1-h exposure to concentrations of thimerosal of 0.0005% or greater, virtually all corneal cells present in established cultures are killed. These results suggest that use of ophthalmic preparations containing these chemicals may affect the metabolic and proliferative capacity of the corneal epithelium adversely.

[Individual sensitivity of cilia. A simple in vitro test method]. / Individuelle Empfindlichkeit der Cilien. Eine einfache in-vitro-Testmethode.

A simple patient-related method is described that determines the effect of toxic substances on the ciliary activity of human nasal cilia cells. The method is suitable for investigating and numerically determining substance-specific toxicity and individual sensitivity even between patients of different states of health. This is illustrated by the example of two preservatives introduced in human medicine: chlorhexidine and merthiolate.