Arsenobetaine as the major arsenic compound in the muscle of two species of freshwater fish

The chemical form of arsenic contained in the muscle of certain freshwater fish was examined using cultured specimens of rainbow trout (Salmo gairdneri) and wild specimens of Japanese smelt (Hypomesus nipponensis). More than 95% of the total arsenic of both species was extracted with methanol and recovered in the water-soluble fraction. The major arsenic compound of both species was purified by cation-exchange chromatography on Dowex 50, gel filtration on Bio-Gel P-2 and HPLC on Asahipak GS-220H. Behavior in the above purification procedure and analyses of the purified compounds by HPLC–ICP and TLC confirmed that the major arsenic compound of both species was arsenobetaine. Arsenobetaine found in cultured rainbow trout seems to be derived from the commercial assorted feed containing arsenobetaine as the major arsenical. On the other hand, the result with wild Japanese smelt suggested that arsenobetaine is a naturally occurring compound in the freshwater environment.     

Identification of arsenic species in sheep‐wool extracts by different chromatographic methods

Sheep on the island of North Ronaldsay (Orkney, UK) feed mostly on seaweed, which contains high concentrations of dimethylated arsenoribosides. Wool of these sheep contains dimethylated, monomethylated and inorganic arsenic, in addition to unidentified arsenic species in unbound and complexed form. Chromatographic techniques using different separation mechanisms and detectors enabled us to identify five arsenic species in water extracts of wool. The wool contained 5.2 ± 2.3 µg arsenic per gram wool. About 80% of the arsenic in wool was extracted by boiling the wool with water. The main species is dimethylarsenic, which accounted for about 75 to 85%, monomethylated arsenic at about 5% and the rest is inorganic arsenic. Depending on the separation method and condition, the chromatographic recovery of arsenic species was between 45% for the anion exchange column, 68% for the size exclusion chromatography (SEC) and 82% for the cation exchange column. The SEC revealed the occurrence of two unknown arsenic compounds, of which one was probably a high molecular mass species. Since chromatographic recovery can be improved by either treating the extract with CuCl/HCl (CAT: 90%) or longer storage of the sample (CAT: 105%), in particular for methylated arsenic species, it can be assumed that labile arsenic–protein‐like coordination species occur in the extract, which cannot be speciated with conventional chromatographic methods. It is clear from our study of sheep wool that there can be different kinds of ‘hidden’ arsenic in biological matrices, depending on the extraction, separation and detection methods used. Hidden species can be defined as species that are not recordable by the detection system, not extractable or do not elute from chromatographic columns. Copyright © 2003 John Wiley & Sons, Ltd.     

Metabolism of arsenic compounds by the blue mussel mytilus edulis after accumulation from seawater spiked with arsenic compounds

Blue mussels (Mytilus edulis) were exposed to 100 μg As dm^?3 in the form of arsenite, arsenate, methylarsonic acid, dimethylarsinic acid, arsenobetaine, arsenocholine, trimethylarsine oxide, tetramethylarsonium iodide or dimethyl-(2-hydroxyethyl)arsine oxide in seawater for 10 days. The seawater was renewed and spiked with the arsenic compounds daily. Analyses of water samples taken 24 h after spiking showed that arsenobetaine and arsenocholine had been converted to trimethylarsine oxide, whereas trimethylarsine oxide and tetramethylarsonium iodide were unchanged. Arsenobetaine was accumulated by mussels most efficienty, followed in efficiency by arsenocholine and tetramethylarsonium iodide. None of the other arsenic compounds was significantly accumulated by the mussels. Extraction of mussel tissues with methanol revealed that control mussels contained arsenobetaine, a dimethyl-(5-ribosyl)arsine oxide and an additional arsenic compound, possibly dimethylarsinic acid. Mussels exposed to arsenobetaine contained almost all their experimentally accumulated arsenic as arsenobetaine, and mussels exposed to tetramethylarsonium iodide contained it as the tetramethylarsonium compound. Mussels exposed to arsenocholine had arsenobetaine as the major arsenic compound and glycerylphosphorylarsenocholine as a minor arsenic compound in their tissues. The results show that arsenobetaine and arsenocholine are efficiently accumulated from seawater by blue mussels and that in both cases the accumulated arsenic is present in the tissues as arsenobetaine. Consequently arsenobetaine and/or arsenocholine present at very low concentrations in seawater may be responsible for the presence of arsenobetaine in M. edulis and probably also among other marine animals. The quantity of arsenobetaine accumulated by the mussels decreases with increasing concentrations of betaine. HPLC-ICP-MS was found to be very powerful for the investigation of the metabolism of arsenic compounds in biological systems.     

Effects of arsenobetaine,a major organic arsenic compound in seafood,on the maturation and functions of human peripheral blood monocytes,macrophages and dendritic cells

We examine the in vitro immunotoxicity of synthetically pure arsenobetaine [AsBe; trimethyl (carboxymethyl) arsonium zwitterion], which is a major organic arsenic compound in seafood, on various human immune cells, such as peripheral blood monocytes, monocyte‐derived macrophages and monocyte‐derived dendritic cells (DCs). In particular, we examine the differentiation of monocytes into macrophages or DCs by comparing the effects of AsBe with those pentavalent inorganic arsenate. AsBe neither enhanced nor inhibited the differentiation of human monocytes into macrophages or DCs, and also did not affect their various immune functions. Furthermore, AsBe had no cytolethality in monocyte‐derived macrophages or DCs even at a concentration of 5 mmol l⁻¹. In contrast, inorganic arsenate showed strong cytolethality in these human immune cells in vitro at micromolar concentrations. These data indicate that the organic arsenic compound AsBe in seafood has no in vitro immunotoxicity in human immune cells. Copyright © 2004 John Wiley & Sons, Ltd.     

The chemical form and acute toxicity of arsenic compounds in marine organisms

A method for the separation and identification of inorganic and methylated arsenic compounds in marine organisms was constructed by using a hydride generation/cold trap/gas chromatography mass spectrometry (HG/CT/GC MS) measurement system. The chemical form of arsenic compounds in marine organisms was examined by the HG/CT/GC MS system after alkaline digestion. It was observed that trimethylarsenic compounds were distributed mainly in the water-soluble fraction of muscle of carnivorous gastropods, crustaceans and fish. Also, dimethylated arsenic compounds were distributed in the water-soluble fraction of Phaeophyceae. It is thought that most of the trimethylated arsenic is likely to be arsenobetaine since this compound released trimethylarsine by alkaline digestion and subsequent reduction with sodium borohydride. The major arsenic compound isolated from the water-soluble fraction in the muscle and liver of sharks was identified as arsenobetaine from IR, FAB Ms data, NMR spectra and TLC behaviour. The acute toxicity of arsenobetaine was studied in male mice. The LD₅₀ value was higher than 10 g kg⁻¹. This compound was found in urine in the non-metabolized form. No particular toxic symptoms were observed following administration. These results suggest that arsenobetaine has low toxicity and is not metabolized in mice. The LD₅₀ values of other minor arsenicals in marine organisms, trimethylarsine oxide, arsenocholine and tetramethylarsonium salt, were also examined in mice.     

Metabolism of methylated arsenic compounds by arsenic-resistant bacteria (Klebsiella oxytoca and Xanthomonas sp.)

Two bacteria exhibiting resistance to toxic arsenic were isolated. These had been contaminated with arsenic in a Chlorella sp. culture medium containing arsenic. The two bacteria were identified as Klebsiella oxytoca and Xanthomonas sp., and grew well in a peptone medium at neutral pH at 30°C, reaching the stationary phase in ca 100h and 70h, respectively. The growth of the bacteria was not affected by arsenic(V) concentrations in the medium as high as 1000mg dm^?3. The bacteria bioaccumulated arsenic, a part of the arsenic being methylated. The bioaccumulation exhibited its peak around the turing point from the log phase to the stationary phase. The relative content of methylated arsenic in the excrement was greater than that in the bacterial cells. Adaptation treatment of inorganic arsenic caused an increase in the bioaccumulation of inorganic arsenic by K. oxytoca. Such a situation was not observed in the case of Xanthomonas sp. The bacteria also bioaccumulated methylated arsenic compounds, and demethylation of these species was observed. When the bacteria were killed by ethanol, arsenic was not taken up by the cells.     

Electrochemical methods for the determination of total arsenic and arsenic compounds

The determination of total arsenic and of arsenic compounds in biological and inorganic samples is a task frequently encountered by analysts. Several elecrochemical methods have been developed for the determination of total arsenic (generally after mineralization of the sample), arsenite, arsenate, methylarsonic acid and dimethylarsinic acid. The electrochemical behavior of several other organic arsenic compounds was also studied. This paper reviews these electrochemical methods, their application to environmental samples, and the problems encountered in the electrochemical determination of arsenic and arsenic compounds.     

利用氢化物发生-冷阱捕集-原子吸收光谱联用技术测定雄黄染毒大鼠血中砷的含量

采用氢化物发生-冷阱捕集-原子吸收光谱法(HG-CT-AAS)测定了雄黄染毒大鼠血中不同形态砷的含量.结果表明,雄黄在大鼠体内的代谢产物为无机砷、一甲基胂酸和二甲基胂酸,其中二甲基胂酸是主要代谢产物;3种形态砷的精密度、准确度、回收率及线性关系良好.     

Decomposition of organoarsenic compounds for total arsenic determination in marine organisms by the hydride generation technique

The conditions necessary for the complete decomposition of six organic arsenic compounds, namely methylarsonic acid (MMAA), dimethylarsinic acid (DMAA), trimethylarsine oxide, tetramethylarsonium iodide, arsenocholine bromide (AsC) and arsenobetaine (AB), were investigated. The degree of decomposition of the arsenic compounds was monitored using a hydride generation (HYD) technique, because the response from this system depends strongly on the chemical species of arsenic, with inorganic arsenic (the expected product from these decomposition experiments) giving a much more intense HYD signal than the organic arsenic compounds. The arsenic compounds were decomposed by heating them with three types of acid mixture, namely HNO₃? HClO₄, HNO₃? HClO₄? HF, or HNO₃? HClO₄? H₂SO₄. Both MMAA and DMAA were decomposed completely using any of the mixed acids at a decomposition temperature of 200 °C or higher. The HNO₃? HClO₄? H₂SO₄ mixture was the most effective for decomposing AsC and AB, which are the most difficult compounds among all types of organic arsenic compound to decompose and render inorganic. The complete decomposition of AB was only achieved, however, when the temperature was 320 °C or higher, and the sample was evaporated to dryness. When the residue from this treatment was examined by high‐performance liquid chromatography combined with inductively coupled plasma atomic emission spectrometry, all of the arsenic was found to be present as arsenic(V). The optimized conditions (HNO₃? HClO₄? H₂SO₄ at 320 °C) for decomposing AB were then used to determine the total amount of arsenic in marine organisms known to contain AB. Copyright © 2005 John Wiley & Sons, Ltd.     

Evaluation of the influence of arsenic species on the nitrogen metabolism of a model angiosperm: nasturtium,Tropaeolum majus

How the various organic and inorganic arsenic species affect the nitrogen metabolism of a model plant, Tropaeolum majus, was studied in order to evaluate the toxicological impact of the various chemical forms of arsenic. For this purpose, the effects on the (a) entire nitrogen pool, (b) protein fraction, and (c) non‐protein fraction were distinguished. The arsenic‐dependent effects on the nitrogen cycle were assessed by using ¹⁵N‐labelled KNO₃ as a nutritive substance and optical emission spectroscopy to analyse how ¹⁵N is incorporated into the nitrogen cycle. In addition to the ¹⁵N‐tracer experiments, the uptake and metabolization of the arsenic compounds were examined. The work shows that biochemical indicator systems like ¹⁵N‐tracer studies are able to characterize the degree of the influence of metabolic processes by arsenic species. For example, the incorporated ¹⁵N concentration decreased linearly and independently of the ¹⁵N fraction with increasing dimethylarsinate (DMA) concentrations. This behaviour indicates that DMA has prevented the uptake of ¹⁵N and hence the formation of amino acids and proteins. Arsenite‐treated plants exhibited an elevated concentration of non‐protein ¹⁵N, which could be an indication either for a stimulated uptake of nitrate or for an interrupted amino acid/protein synthesis. Copyright © 2005 John Wiley & Sons, Ltd.     

Lipophilic arsenic compound(s) in the liver of a tiger shark (Galeocerdo cuvier)

Homogenized aliquots (100 g) of the liver (8.4 kg, 5 m?g As g^?1) of a tiger shark (Galeocerdo cuvier) were extracted with chloroform/methanol, and the extracts purified by countercurrent extraction (hexane/87% ethanol), silica gel column chromatography (chloroform/methanol mixtures as mobile phases), and silica gel thin-layer chromatography (chloroform/methanol/acetic acid). The purified samples (24 mg arsenic g^?1) gave no ³¹P NMR signal, but gave ¹H and ¹³C NMR signals with similarities to those of dipalmitoylphosphatidic acid and salad on and also signals indicative of the presence of methylated arsenic compounds. The sample could contain a diacyl glyceride with a methylated arsenic group.     

Chemical fingerprint of Hoodia species,dietary supplements,and related genera by using HPTLC

A HPTLC method was developed for simple and rapid chemical fingerprint analysis of four Hoodia species, dietary supplements that claim to contain Hoodia gordonii, and plants from genera related to Hoodia. HPTLC was performed on precoated silica 60F₂₅₄ plates with dichloromethane/methanol/water 75:17:2.2 by volume, as mobile phase. Evaluation of the HPTLC plates was done by using the CAMAG DigiStore2 digital system with winCATS software. The authentication of H. gordonii was achieved by comparing the band colors and R_f values for TLC fingerprints with those of 11 standard compounds including P57. The developed method was successfully applied for the identification of the 11 pregnane glycosides for four different species of Hoodia, 24 related genera and 13 dietary supplements that claim to contain H. gordonii. Different sample matrices were successfully analyzed, providing a wide range of applicability for this method, including gels, capsules, tablets, sprays, teas, snack bars, powders, and juices. The developed method was validated for specificity, stability, repeatability, and robustness. The results of HPTLC method were verified by LC‐UV‐MS method.     

Determination of arsenic species in marine organisms by HPLC-ICP-OES and HPLC-HG-QFAAS

Separation and quantification of six arsenic species have been performed in cod, tuna and mussel samples by high performance liquid chromatography (HPLC) using inductively coupled plasma-optical emission spectrometry (ICP-OES) and hydride generation-quartz furnace atomic absorption spectrometry (HG-QFAAS) as detection techniques. It has been shown that arsenic extraction with a water-methanol (11) mixture is sufficiently quantitative for the cod and tuna, in which arsenic is mainly present as arsenobetaine (about 90% of total As extracted). In contrast, only 60% of the element is extracted from the mussels and the chromatograms obtained reveal the presence of an unknown compound. Detection limits are in the g ml^–1 range for the HPLC-ICP-OES technique (quantification of arsenobetaine and arsenocholine) and in the ng ml^–1 range for the HPLC-HG-QFAAS system (quantification of arsenite, arsenate, monomethylarsonic and dimethylarsinic acids).     

Distribution and cycle of arsenic compounds in the ocean

In order to understand the distribution and the cycle of arsenic compounds in the marine environment, the horizontal distributions of arsenic(V) [As(V)], arsenic(III) [As(III)], monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA) in the Indian Pacific Oceanic surface waters have been investigated. This took place during cruises of the boat Shirase from Tokyo to the Syowa Station (15 November–19 December 1990), of the tanker Japan Violet from Sakai to Fujayrah (28 July–17 August 1991) and of the boat Hakuho-maru from Tokyo to Auckland (19 September–27 October 1992). Vertical distributions of arsenic in the west Pacific Ocean have also been investigated. The concentration of As(V) was found to be relatively higher in the Antarctic than in the other areas. Its concentration varied from 340 ng dm^?3 (China Sea) to 1045 ng dm^?3 (Antarctic). On the other hand, the concentrations of the biologically produced species, MMAA and DMAA, were extremely low in the Antarctic and southwest Pacific waters. Their concentrations in Antarctic waters were 8 ng dm^?3 and 22 ng dm^?3 and those in the southwest Pacific were 12 ng dm^?3 and 25 ng dm^?3. In the other regions the concentration varied from 16 ng dm^?3 (China Sea) to 36 ng dm^?3 (north Indian Ocean) for MMAA and from 50 ng dm^?3 (east Indian Ocean) to 172 ng dm^?3 (north Indian Ocean) for DMAA. As a result, with the exception of Antarctic and southwest Pacific waters, the percentages of each arsenic species in the surface waters were very similar and varied from 52% (east Indian Ocean) to 63% (northwest Pacific Ocean) for As(V), from 22% (northwest Pacific Ocean) to 27% (east Indian Ocean) for As(III) and from 15% (northwest Pacific Ocean) to 21% (north and east Indian Oceans) for the methylated arsenics (MMAA+DMAA). These percentages in Antarctic waters were 97%, 0.2% and 2.8%, respectively, and those in the southwest Pacific Ocean were 97% for As(V)+As(III) and 3% for MMAA+DMAA. The very low concentrations of the biologically produced species in Antarctic waters and that of methylated arsenic in southwest Pacific waters indicated that the microorganism communities in these oceans was dominated by microorganisms having a low affinity towards arsenic. Furthermore, microorganism activity in the Antarctic was also limited due to the much lower temperature of the seawater there. The vertical profile of inorganic arsenic was 1350 ng dm^?3 in surface waters, 1500 ng dm^?3 in bottom waters with a maximum value of 1700 ng dm^?3 at a depth of about 2000 m in west Pacific waters. This fact suggested the uptake of arsenic by microorganisms in the surface waters and the co-precipitation of arsenic with hydrated heavy-metal oxides in bottom waters. The suggested uptake of inorganic arsenic and subsequent methylation was also supported by the profile of DMAA, with a high concentration of about 26 ng dm^?3 in surface water and a significant decrease to a value of 9 ng dm^?3 at a depth of 1000 m.     

Uptake and degradation of arsenobetaine by the microorganisms occurring in sediments

We have reported the degradation of arsenobetaine [(CH₃)₃As⁺CH₂COO^?] to inorganic arsenic by microorganisms from various marine origins such as sediments. However, there was no information as to the fate of the ingested arsenobetaine within the body of the microorganisms before excretion. In this study, arsenobetaine and sediments were added to two culture media (1/5 Zobell 2216E and a solution of inorganic salts) and aerobically incubated at 25°C in the dark. Despite the degradation and complete disappearance of arsenobetaine from the filtrates of the incubation mixtures, the major arsenic compound from the microorganisms harvested from the mixtures was identified by HPLC as arsenobetaine throughout the incubation period. The presence of arsenobetaine was further confirmed by TLC and fast atom bombardment mass spectrometry (FAB MS). A minor arsenical also present in the incubated microorganisms, dimethylarsinic acid, was detected.     

Separation of arsenic species by ion-pair and ion exchange high performance liquid chromatography

Arsenite, arsenate, monomethylarsonate, dimethylarsinate, arsenobetaine, arsenocholine and the tetramethylarsonium ion were subjected to ion-exchange and ion-pair reversed phase HPLC. The ion exchange method was superior in selectivity and time of analysis for the arsenic anions. The ammonium ions used for the ion-pair method only resulted in separation of some of the anionic arsenic compounds. Flame atomic absorption spectrometry was used for on-line arsenic-specific detection.     

云南阳宗海砷污染水平、变化趋势及风险评估

云南阳宗海砷污染事件引起社会广泛关注.为了解事件发生后阳宗海砷污染水平及变化趋势,分别于2008年12月、2009年2月、5月及9月四次采样,研究了阳宗海湖水、底泥、周边井水、土壤、农作物及水生生物中的砷含量及其变化趋势.研究结果显示:湖水平均砷浓度分别为176.9、147.3、159.3和161.1μg/L(算术平均),底泥平均浓度分别为32.87、62.41、62.99和46.96μg/g(算术平均).阳宗海湖水砷浓度经历了先升后降再到平稳的变化过程,底泥砷含量迅速升高后缓慢下降,湖水和底泥间砷交换还在进行.阳宗海附近土壤中砷最高浓度为23.33μg/g,未超过国家土壤环境质量三级标准.大米、玉米、花椰菜、小油菜等农作物可食用部分中砷的最高值为0.35μg/g,均未超过国家无公害食品标准.水生植物中砷水平大多在100～200μg/g之间,最高为苦草,砷含量超过300μg/g,说明该植物对砷有一定的富集能力.虾、鱼类等可食用水生动物砷浓度范围为1.52～11.4μg/g.     

Pulmonary toxicity of indium arsenide and arsenic selenide following repeated intratracheal instillations to the lungs of hamsters

Chronic toxicity of indium arsenide (InAs) and arsenic selenide (As₂Se₃) was studied in male Syrian golden hamsters which received InAs or As₂Se₃ particles, each containing a total dose of 7.5 mg of arsenic, by intratracheal instillations once a week for 15 weeks. As a control, hamsters were treated with the vehicle, phosphate buffer solution. During their total lifespan, the cumulative body weight gain of the hamsters in the InAs group was suppressed significantly compared with that in the control group, but not in the As₂Se₃ group when compared with that in the control group. However, the survival rate for the InAs group was significantly higher compared with the control group, but not for the As₂Se₃ group when compared with the control group. During the animals' total lifespan, one lung adenoma was seen in the 27 hamsters in the InAs group and one lung adenoma in the 23 hamsters in the control group. No tumors of the lung were observed in the As₂Se₃ group. Malignant tumors outside the lung appeared in four hamsters in the InAs group and in two in the As₂Se₃ group. No non-lung malignant tumours were seen in the control group. Total tumor incidence rates were 25.9% (7/27) in the InAs group, 10.3% (3/29) in the As₂Se₃ group and 8.7% (2/23) in the control group. There were therefore no significant differences in tumor incidence between the InAs or the As₂Se₃ group, and the control group. Regarding histopathological findings in the lung, incidence rates of proteinosis-like lesions, pneumonia, metaplastic ossification and emphysema were seen only in the InAs group, and alveolar or bronchiolar cell hyperplasia observed in both the InAs and the As₂Se₃ groups were at significantly higher rates than those in the control group. From these results, it was concluded that InAs and As₂Se₃ particles could induce pulmonary toxicity when instilled intratracheally into hamsters. A great deal of attention should be paid to the toxicity of both InAs and As₂Se₃, even though in this study the adverse health effects of As₂Se₃ appeared to be less than those of InAs.     

Bioaccumulation and excretion of arsenic compounds by a marine unicellular alga,polyphysa peniculus

Polyphysa peniculus was grown in artificial seawater in the presence of arsenate, arsenite, monomethylarsonate and dimethylarsinic acid. The separation and identification of some of the arsenic species produced in the cells as well as in the growth medium were achieved by using hydride generation–gas chromatography–atomic absorption spectrometry methodology. Arsenite and dimethylarsinate were detected following incubation with arsenate. When the alga was treated with arsenite, dimethylarsinate was the major metabolite in the cells and in the growth medium; trace amounts of monomethylarsonate were also detected in the cells. With monomethylarsonate as a substrate, the metabolite is dimethylarsinate. Polyphysa peniculus did not metabolize dimethylarsinic acid when it was used as a substrate. Significant amounts of more complex arsenic species, such as arsenosungars, were not observed in the cells or medium on the evidence of flow injection–microwave digestion–hydride generation–atomic absorption spectrometry methodology. Transfer of the exposed cells to fresh medium caused release of most cell–associated arsenicals to the surrounding environment.     

Interpretation of inorganic arsenic metabolism in humans in the light of observations made in vitro and in vivo in the rat

The toxicity of inorganic trivalent arsenic for living organisms is reduced by in vivo methylation of the element. In man, this biotransformation leads to the synthesis of monomethylarsonic (MMA) and dimethylarsinic (DMA) acids, which are efficiently eliminated in urine along with the unchanged form (As_i). In order to document the methylation process in humans, the kinetics of As_i, MMA and DMA elimination were studied in volunteers given a single dose of one of these three arsenicals or repeated doses of As_i. The arsenic methylation efficiency was also assessed in subjects acutely intoxicated with arsenic trioxide (As₂O₃) and in patients with liver diseases. Several observations in humans can be explained by the properties of the enzymic systems involved in the methylation process which we have characterized in vitro and in vivo in rats as follows: (1) production of As_i metabolites is catalyzed by an enzymic system whose activity is highest in liver cytosol; (2) different enzymic activities, using the same methyl group donor (S-adenosylmethionine), lead to the production of mono- and di-methylated derivatives which are excreted in urine as MMA and DMA; (3) dimethylating activity is highly sensitive to inhibition by excess of inorganic arsenic; (4) reduced glutathione concentration in liver moderates the arsenic methylation process through several mechanisms, e.g. stimulation of the first methylation reaction leading to MMA, facilitation of As_i uptake by hepatocytes, stimulation of the biliary excretion of the element, reduction of pentavalent forms before methylation, and protection of a reducing environment in the cells necessary to maintain the activity of the enzymic systems.