Arsenic accumulation in livers of pinnipeds, seabirds and sea turtles: subcellular distribution and interaction between arsenobetaine and glycine betaine

Concentrations of total arsenic and individual arsenic compounds were determined in liver samples of pinnipeds (northern fur seal Callorhinus ursinus and ringed seal Pusa hispida), seabirds (black-footed albatross Diomedea nigripes and black-tailed gull Larus crassirostris) and sea turtles (hawksbill turtle Eretmochelys imbricata and green turtle Chelonia mydas). Among these species, the black-footed albatross contained the highest hepatic arsenic concentration (5.8+/-3.7 microg/g wet mass). Arsenobetaine was the major arsenic species found in the liver of all these higher tropic marine animals. To investigate the cause of high accumulation of arsenobetaine, subcellular distribution of arsenic and relationship between arsenobetaine and glycine betaine concentrations were examined in the livers of these animals. There was no relationship between total arsenic concentration and its subcellular distribution in liver tissues. However, a significant negative correlation was found between arsenobetaine and glycine betaine concentrations in the liver of six species examined. This result may indicate that arsenobetaine is accumulated in these marine animals as an osmolyte along with glycine betaine, which is a predominant osmolyte in marine animals because the chemical structure and properties of arsenobetaine are similar to those of glycine betaine.     

The degradation of arsenobetaine to inorganic arsenic by sedimentary microorganisms

Two growth media containing arsenobetaine [(CH₃)₃ As⁺ CH₂COO^–] were mixed with coastal marine sediments, the latter providing a source of microorganisms. The mixtures were kept at 25 °C in the dark and shaken for several weeks under an atmosphere of air. The disappearance of arsenobetaine and the appearance of two metabolites were followed by HPLC. The HPLC-retention time of the first metabolite agreed with that of trimethylarsine oxide [(CH₃)₃AsO]. The second metabolite was identified as arsenate (As(V)) using hydride generation/cold trap/GC MS analysis and thin layer chromatography. This is the first scientific evidence showing that arsenobetaine is degraded by microorganisms to inorganic arsenic via trimethylarsine oxide. The degradation of arsenobetaine to inorganic arsenic completes the marine arsenic cycle that begins with the methylation of inorganic arsenic on the way to arsenobetaine.     

Gene-mutation induction by arsenic compounds in the mouse lymphoma assay

Arsenic compounds are generally considered as poor inducers of gene mutations. To investigate the mutagenicity of several arsenic compounds at the thymidine kinase (Tk) gene, a reporter gene for mutation induction, we used the mouse lymphoma assay (MLA). This test is widely applied and detects a broad spectrum of mutational events, from point mutations to chromosome alterations. The selected arsenic compounds were two inorganic (sodium arsenite and arsenic trioxide) and four organic compounds (monomethylarsonic acid, dimethylarsinic acid, tetraphenylarsenium and arsenobetaine). The results show that sodium arsenite, arsenic trioxide, monomethylarsonic acid and dimethylarsinic acid are mutagenic, showing a clear dose-response pattern. On the other hand, tetraphenylarsenium and arsenobetaine are not mutagenic. Inorganic arsenic compounds are the more potent agents producing significant effects in the micromolar range, while the mutagenic organic arsenic compounds induce similar effects but in the millimolar range.     

Gene-mutation induction by arsenic compounds in the mouse lymphoma assay

Arsenic compounds are generally considered as poor inducers of gene mutations. To investigate the mutagenicity of several arsenic compounds at the thymidine kinase (Tk) gene, a reporter gene for mutation induction, we used the mouse lymphoma assay (MLA). This test is widely applied and detects a broad spectrum of mutational events, from point mutations to chromosome alterations. The selected arsenic compounds were two inorganic (sodium arsenite and arsenic trioxide) and four organic compounds (monomethylarsonic acid, dimethylarsinic acid, tetraphenylarsenium and arsenobetaine). The results show that sodium arsenite, arsenic trioxide, monomethylarsonic acid and dimethylarsinic acid are mutagenic, showing a clear dose–response pattern. On the other hand, tetraphenylarsenium and arsenobetaine are not mutagenic. Inorganic arsenic compounds are the more potent agents producing significant effects in the micromolar range, while the mutagenic organic arsenic compounds induce similar effects but in the millimolar range.     

Uptake and elimination of arsenobetaine by the mussel Mytilus edulis is related to salinity

The high concentrations of the naturally occurring arsenic compound arsenobetaine in marine animals, in comparison with freshwater animals, has led to the suggestion that salinity is a factor in its accumulation. In separate experiments, we investigated the uptake and elimination of arsenobetaine by the mussel Mytilus edulis when maintained under three salinity regimes (32, 24, and 16 practical salinity units). Both uptake and elimination of arsenobetaine depended on the salinity of the water in a manner leading to higher concentrations at the higher salinity. The data are consistent with a proposed role of arsenobetaine as an adventitiously acquired osmolyte, and readily explain field data for freshwater and marine animals.     

Uptake of arsenic-betaines by the mussel Mytilus edulis

Mussels (Mytilus edulis) were exposed to trimethyl(carboxymethyl)arsonium bromide (arsenobetaine, C-1 betaine), trimethyl(2-carboxyethyl)arsonium bromide (C-2 betaine), or trimethyl(3-carboxypropyl)arsonium bromide (C-3 betaine). Arsenic was accumulated by the mussels in all cases but the efficiency of uptake decreased with the number of methylene units in the carboxyalkyl group. Arsenobetaine (C-1 betaine) was the most readily accumulated, followed by the C-2 betaine (70% as efficient as arsenobetaine) and the C-3 betaine (∼7%). Chromatographic analysis (HPLC-ICPMS) of extracts of the mussels demonstrated that the arsenic compounds were accumulated uncahanged. A 46-day depuration period which followed exposure did not significantly reduce the arsenic concentration in any of the three groups. Comparison with previous data on accumulation of arsenic compounds by M. edulis indicates that uptake may be influenced by the presence of a quaternary arsonium group and the zwitterionic nature of the arsenic-betaines.     

Degradation of arsenobetaine to inorganic arsenic by bacteria in seawater

The substances suspended in seawater were fractionated by membrane filtration into three fractions. Fraction 1 was collected on a membrane filter of 0.22 µm pore-size, fraction 2 on a 5 µm pore-size and fraction 3 on 0.22 µm pore-size from the filtrate passed through the 5 µm membrane filter. Arsenobetaine was incubated with each of these fractions in two media (ZoBell 2216E and a solution of inorganic salts) at 25 °C in the dark under aerobic conditions. The mixture added with fraction 3 was considered to contain only bacteria. In every case, in the inorganic salt medium, inorganic arsenic(V) was derived from arsenobetaine via trimethylarsine oxide. In the ZoBell medium, arsenobetaine was not degraded to inorganic arsenic, although trimethylarsine oxide was derived in every case. We conclude that the degradation of arsenobetaine to trimethylarsine oxide or inorganic arsenic can be accomplished in seawater by bacteria alone.     

Arsenic accumulation in three species of sea turtles

Arsenic in the liver, kidney and muscle of three species of sea turtles, e.g., green turtles (Chelonia mydas), loggerhead turtles (Caretta caretta) and hawksbill turtles (Eretmochelys imbricata), were determined using HG-AAS, followed by arsenic speciation analysis using HPLC-ICP-MS. The order of arsenic concentration in tissues was muscle > kidney > liver. Unexpectedly, the arsenic concentrations in the hawksbill turtles feeding mainly on sponges were higher than the two other turtles primarily eating algae and mollusk which accumulate a large amount of arsenic. Especially, the muscles of the hawksbill turtles contained remarkably high arsenic concentrations averaging 153 mg kg^–1 dry weight with the range of 23.1–205 mg kg^–1 (n=4), even in comparison with the data from other organisms. The arsenic concentrations in the tissues of the green turtles were significantly decreased with standard carapace length as an indicator of growth. In arsenic compounds, arsenobetaine was mostly detected in the tissues of all the turtles. Besides arsenobetaine, a small amount of dimethylarsinic acid was also observed in the hawksbill turtles.     

An origin for arsenobetaine involving bacterial formation of an arsenic-carbon bond

Lysed-cell extract of a Pseudomonas sp. was shown to catalyse bioconversion of dimethylarsinoylacetate to arsenobetaine and dimethylarsinate. Provision of the universal methyl donor S-adenosylmethionine to bioconversion mixtures promoted both the rate and extent of arsenobetaine formation. These findings suggest that in the proposed biosynthesis of arsenobetaine from dimethylarsinoylethanol, oxidation (i.e. the formation of the carboxymethyl group of dimethylarsinoylacetate) would precede the reduction and methylation at the arsenic atom. The presence of enzyme(s) capable of methylating dimethylarsinoylacetate in a bacterial isolate from marine mussel (Mylitus edulis), highlights a possible direct involvement of prokaryotic organisms in the biosynthesis of organoarsenic compounds within marine animals.     

Identification of arsenobetaine as a major arsenic compound in muscle of two demersal sharks, shortnose dogfish Squalus brevirostris and starspotted shark Mustelus manazo

The major water-soluble arsenic compound was isolated from the muscle of shortnose dogfish Squalus brevirostris and of starspotted shark Mustelus manazo, both of which are demersal sharks. The isolated compound was identified to be arsenobetaine by its chromatographic and spectrometric analyses.     

Arsenic speciation in human urine reference materials using high-performance liquid chromatography with inductively coupled plasma mass spectrometric detection.

Six arsenic compounds including arsenocholine, arsenobetaine, dimethylarsinic acid, methylarsonic acid, arsenous acid and arsenic acid were separated by high-performance liquid chromatography (HPLC) on a Hamilton PRP-X100 anion-exchange column using isocratic elution and detected by inductively coupled plasma mass spectrometry (ICP-MS). This analytical procedure was applied to the speciation of arsenic compounds in human urine. The influence of urine matrix on the separation of arsenic compounds was evaluated and the determination of arsenic compounds was not hampered by the ArCl interference which has often been encountered in ICP-MS. Three human urine reference materials, SRM 2670 normal level, SRM 2670 elevated level and Lyphocheck urine metal control 1, were analyzed with respect to arsenic compounds by HPLC-ICP-MS. The results were found to be in good agreement with the certified total arsenic concentration in the reference materials. Six arsenic compounds were detected. Arsenobetaine was found to be present in all of the investigated human urine reference materials.     

Arsenic in Seafood: Speciation Issues for Human Health Risk Assessment

Major sources of arsenic exposure for humans are foods, particularly aquatic organisms, which are called seafood in this report. Although seafood contains a variety of arsenicals, including inorganic arsenic, which is toxic and carcinogenic, and arsenobetaine, which is considered nontoxic, the arsenic content of seafood commonly is reported only as total arsenic. A goal of this literature survey is to determine if generalizable values can be derived for the percentage of total arsenic in seafood that is inorganic arsenic. Generalizable values for percent inorganic arsenic are needed for use as default values in U.S. human health risk assessments of seafood from arsenic-contaminated sites. Data from the worldwide literature indicate the percent of inorganic arsenic in marine/estuarine finfish does not exceed 7.3% and in shellfish can reach 25% in organisms from presumably uncontaminated areas, with few data available for freshwater organisms. However, percentages can be much higher in organisms from contaminated areas and in seaweed. U.S. site-specific data for marine/estuarine finfish and shellfish are similar to the worldwide data, and for freshwater finfish indicate that the average percent inorganic arsenic is generally < 10%, but ranges up to nearly 30%. Derivation of nationwide defaults for percent inorganic arsenic in fish, shellfish, and seaweed collected from arsenic-contaminated areas in the United States is not supported by the surveyed literature.     

Arsenic in the human food chain,biotransformation and toxicology – Review focusing on seafood arsenic

Fish and seafood are main contributors of arsenic (As) in the diet. The dominating arsenical is the organoarsenical arsenobetaine (AB), found particularly in finfish. Algae, blue mussels and other filter feeders contain less AB, but more arsenosugars and relatively more inorganic arsenic (iAs), whereas fatty fish contain more arsenolipids. Other compounds present in smaller amounts in seafood include trimethylarsine oxide (TMAO), trimethylarsoniopropionate (TMAP), dimethylarsenate (DMA), methylarsenate (MA) and sulfur-containing arsenicals. The toxic and carcinogenic arsenical iAs is biotransformed in humans and excreted in urine as the carcinogens dimethylarsinate (DMA) and methylarsonate (MA), producing reactive intermediates in the process. Less is known about the biotransformation of organoarsenicals, but new insight indicates that bioconversion of arsenosugars and arsenolipids in seafood results in urinary excretion of DMA, possibly also producing reactive trivalent arsenic intermediates. Recent findings also indicate that the pre-systematic metabolism by colon microbiota play an important role for human metabolism of arsenicals. Processing of seafood may also result in transformation of arsenicals.     

Transport of arsenolipids to the milk of a nursing mother after consuming salmon fish

ObjectiveWe address two questions relevant to infants’ exposure to potentially toxic arsenolipids, namely, are the arsenolipids naturally present in fish transported intact to a mother’s milk, and what is the efficiency of this transport.MethodsWe investigated the transport of arsenolipids and other arsenic species present in fish to mother’s milk by analyzing the milk of a single nursing mother at 15 sampling times over a 3-day period after she had consumed a meal of salmon. Total arsenic values were obtained by elemental mass spectrometry, and arsenic species were measured by HPLC coupled to both elemental and molecular mass spectrometry.ResultsTotal arsenic increased from background levels (0.1 μg As kg⁻¹) to a peak value of 1.72 μg As kg⁻¹ eight hours after the fish meal. The pattern for arsenolipids was similar to that of total arsenic, increasing from undetectable background levels (< 0.01 μg As kg⁻¹) to a peak after eight hours of 0.45 μg As kg⁻¹. Most of the remaining total arsenic in the milk was accounted for by arsenobetaine. The major arsenolipids in the salmon were arsenic hydrocarbons (AsHCs; 55 % of total arsenolipids), and these compounds were also the dominant arsenolipids in the milk where they contributed over 90 % of the total arsenolipids.ConclusionsOur study has shown that ca 2–3 % of arsenic hydrocarbons, natural constituents of fish, can be directly transferred unchanged to the milk of a nursing mother. In view of the potential neurotoxicity of AsHCs, the effects of these compounds on the brain developmental stage of infants need to be investigated.     

Arsenic and Arsenic Species in Cultured Oyster (Crassostrea gigas and C. corteziensis) from Coastal Lagoons of the SE Gulf of California, Mexico

The aim of this study was to evaluate the bioavailability of arsenic (As) through cultured oyster Crassostrea gigas and Crassostrea corteziensis from four coastal lagoons (SE Gulf of California). Organisms were collected in two seasons (rainy and dry season), and they were analyzed for total arsenic and chemical speciation of this element. The concentrations of As in oyster soft tissue fluctuated between 5.44 and 9.56 μg/g for rainy season and 6.46 and 8.33 μg/g for dry season (dry weight) in C. gigas. In C. corteziensis, the As concentrations were <5 μg/g for both seasons (dry weight). Arsenic speciation indicated arsenobetaine as the major arseno-compound accounting for 43.2–76.3 % of total content of As. Lower contributions were obtained for non-extractable As (11.3–17.5 %) and other molecules such as arsenocholine and methyl-arsonate (<5 %). Inorganic arsenic was detectable in only two samples, at concentrations lower than <0.1 μg/g. These As data are the first generated for these mollusks in NW Mexico and indicate that C. gigas and C. corteziensis farmed in this area are safe for human consumption in terms of arseno-compounds.     

Biotransformation of arsenobetaine by microorganisms from the human gastrointestinal tract

Abstract

The consumption of fish and shellfish is a major route of human exposure to arsenic (As), because they contain relatively large concentrations of organoarsenicals, in particular arsenobetaine (AB). AB is considered non-toxic because of its rapid excretion from the human body. However, previous studies on human metabolism and excretion of AB have used the compound in solution rather than considering the effects that occur during the digestion of food in the gastrointestinal tract. In this preliminary study, we used microcosms inoculated with human faecal matter to investigate the aerobic and anaerobic degradation of AB by microorganisms associated with the large intestine. Samples were recovered over 30 days, centrifuged, filtered and the supernatant analysed for total As content and As speciation, using ICP–MS and HPLC–ICP–MS respectively. After 7 days the total As in the supernatants from the aerobic experiment fell to a minimum of 65% of the total added, recovering to 15% less than added after 30 days. By using anion and cation exchange chromatography coupled to ICP–MS detection, arsenobetaine (AB), dimethylarsinic acid (DMA), dimethylarsinoylacetic acid (DMAA) and trimethylarsine oxide (TMAO) were identified as degradation products. Results from the aerobic system showed that after 7 days incubation the AB had been degraded to DMA, DMAA and TMAO and after 30 days the degraded AB reappeared in the samples. The results for the anaerobic system showed no degradation of AB over the 30 day course of the experiment. These findings demonstrate for the first time that biocatalytic capability for AB degradation exists within the human gastrointestinal tract.     

Disposition of arsenobetaine in two marine fish species following administration of a single oral dose of [14C]arsenobetaine

The distribution and excretion of arsenobetaine in fish were investigated using whole body autoradiography and liquid scintillation counting. A single dose of synthesised [(14)C]arsenobetaine was orally administered to Atlantic salmon, Salmo salar L., and Atlantic cod, Gadus morhua L. Arsenobetaine was distributed to most organs within both species. Nevertheless, there were species differences in tissue distribution and excretory pattern. The highest level of arsenobetaine in Atlantic salmon was present in muscle tissue, while high levels of arsenobetaine were found in both muscle and liver (including gall bladder) from Atlantic cod. The results suggest that the major route of excretion was via urine, which seemed to be more important in Atlantic cod than in Atlantic salmon. Elimination of arsenobetaine via bile appeared to be negligible in both species.     

Bacterial degradation of arsenobetaine via dimethylarsinoylacetate

Microorganisms from Mytilus edulis (marine mussel) degraded arsenobetaine, with the formation of trimethylarsine oxide, dimethylarsinate and methylarsonate. Four bacterial isolates from these mixed-cultures were shown by HPLC/hydride generation-atomic fluorescence spectroscopy (HPLC/HG-AFS) analysis to degrade arsenobetaine to dimethylarsinate in pure culture; there was no evidence of trimethylarsine oxide formation. Two of the isolates ( Paenibacillus sp. strain 13943 and Pseudomonas sp. strain 13944) were shown by HPLC/inductively coupled plasma-mass spectrometry (HPLC/ICPMS) analysis to degrade arsenobetaine by initial cleavage of a methyl-arsenic bond to form dimethylarsinoylacetate, with subsequent cleavage of the carboxymethyl-arsenic bond to yield dimethylarsinate. Arsenobetaine biodegradation by pure cultures was biphasic, with dimethylarsinoylacetate accumulating in culture supernatants during the culture growth phase and its removal accompanying dimethylarsinate formation during a carbon-limited stationary phase. The Paenibacillus sp. also converted exogenously supplied dimethylarsinoylacetate to dimethylarsinate only under carbon-limited conditions. Lysed-cell extracts of the Paenibacillus sp. showed constitutive expression of enzyme(s) capable of arsenobetaine degradation through methyl-arsenic and carboxymethyl-arsenic bond cleavage. The work establishes the capability of particular bacteria to cleave both types of arsenic-carbon bonds of arsenobetaine and demonstrates that mixed-community functioning is not an obligate requirement for arsenobetaine biodegradation.     

Arsenobetaine in Atlantic salmon (Salmo salar L.): influence of seawater adaptation

Glycine betaine has been suggested to improve the maintenance of ionic and osmotic homeostasis during seawater adaptation in teleost fish. Arsenobetaine may also behave as an osmolyte, due to its structural similarity to glycine betaine. The influence of seawater adaptation on intestinal uptake and muscle accumulation of arsenobetaine in the teleost Atlantic salmon (Salmo salar L.) was investigated. Atlantic salmon (freshwater and seawater adapted) were given a single oral dose of arsenobetaine, which was absorbed over the intestine within 6 h after exposure. Seawater adapted Atlantic salmon had significantly higher levels of accumulated arsenobetaine in blood compared to the freshwater adapted salmon. However, seawater adaptation had no effect on the levels of accumulated arsenobetaine in muscle tissue. Similar retention of the administered dose was found in muscle tissue in both freshwater and seawater adapted salmon, with 49+/-6% and 50+/-10% retention after 144 h, respectively. Results indicate that muscle retention was not influenced by salinity in seawater adapting teleosts.     

A Novel Kauranoid Isolated from Gibberella fujikuroi

A novel arseno-sugar was isolated from the brown alga Sargassum thunbergii. Instead of the dimethylarsinoyl group reported for algal arseno-sugars, this has a tri-methylarsonium group, which is borne by arsenobetaine, a ubiquitous organoarsenic compound in marine animals. This may be an intermediate between arseno-sugars and arsenobetaine.