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Microbial Methylation of Metalloids: Arsenic, Antimony, and Bismuth

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260,¹ Department of Chemistry, Sam Houston State University, Huntsville, Texas 77341-2117²

SUMMARY

INTRODUCTION

Biomethylation and Bioalkylation

Methylation of metalloids.

Early history.

ARSENIC FUNGI

Wallpaper: Hazardous to Health

Work of Bartolomeo Gosio

Microbiological Test for Arsenic

WORK OF CHALLENGER AND ASSOCIATES (''LEEDS SCHOOL'')

Gosio Gas Is Trimethylarsine

Challenger Mechanism for Arsenic Biomethylation

Role of SAM

ARSENIC METHYLATION

Fungi and Yeasts

Bacteria

Role of Anaerobic Microorganisms

Methyl Donor in Anaerobes

ENZYMOLOGY OF BIOMETHYLATION

Enzymes Catalyzing Reductions

Arsenate reductase.

Methylarsonate reductase.

Enzymes Catalyzing Methyl Group Transfers

ARSENIC BIOMETHYLATION AS DETOXIFICATION?

BIOMETHYLATION OF ANTIMONY

Introduction

Antimony and SIDS

Microbial Methylation of Antimony

METHYLATION OF BISMUTH

EPILOGUE

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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The first observation of a biological methylation (16) came when His, with an interest in the detoxification of aromatic compounds, administered pyridine to a dog (122). N-Methylpyridine was excreted in the urine. Similar examples of the methylation of aromatic compounds were the conversion, xanthine methylxanthine in rabbits, and nicotinic acid trigonelline in dogs (2).

Much later, nutritional experiments showing, for example, that rats could substitute homocystine for methionine in the diet if choline was present led to the concept of transmethylation. This was defined initially as the transfer of a methyl group from one compound to form another N-methyl or S-methyl compound (80). A very important role for methionine, a methylated sulfur compound (111), was slowly recognized, and S-adenosylmethionine (SAM) was identified as the product of the enzymatic activation of methionine in transmethylation reactions (206). The role of SAM as the methyl donor in hundreds of methylation reactions is now well established (36, 37). The mechanism for the de novo biosynthesis of methyl groups has also been determined (90).

Methylation of metalloids. The C, O, N, and S atoms of organic compounds frequently function as methyl group acceptors in primary and secondary metabolic processes (37, 206). This article concerns another phenomenon, the use of metalloids as methyl group acceptors with the major emphasis on the production of volatile compounds by microorganisms. Because of its widespread distribution and many uses in agriculture, industry and medicine, most of the research has concerned arsenic. Like nitrogen, arsenic is a member of group 15 of the periodic table (International Union of Pure and Applied Chemistry recommendation). Work with other members of this group, antimony and bismuth, will also be included. It is of interest that antimony and arsenic share some chemical and toxicological properties (96). Microbial biosynthesis of methylated metalloids is now of considerable academic interest, and the physiological actions of the methylated products are of concern in disciplines such as medicine, toxicology and environmental studies.

The term "biomethylation" describes the formation of both volatile and nonvolatile methylated compounds of metals and metalloids. For arsenic, the major volatile compounds formed by biomethylation have the structure, (CH₃)_nAsH_3-n; for n = 1, 2, and 3, the products are mono-, di-, and trimethylarsine, respectively. The major nonvolatile compounds are methylarsonate and dimethylarsinate. The nomenclature of the arsenic oxyacids is somewhat confusing (Table 1). Potassium arsenite preparations have a variable composition; the commercial article corresponding approximately to KAsO₂ · HAsO₂. The "meta-acid" HAsO₂ (i.e., H₃AsO₃ minus H₂O) and its salts are not known in solution. Acids will generally be referred to as ionized forms (e.g., arsenate for arsenic acid), since these are presumably present under physiological conditions.

The term "bioalkylation" has sometimes been used for more-complex naturally occurring materials, for instance, trimethylarsonium betaine, (CH₃)₃As⁺CH₂COO^-, discovered originally in Australian rock lobster (Panulirus longipes cygnus); lipoidal substances such as O-phosphatidyl trimethylarsoniumlactic acid (Fig. 1A), found in a marine diatom; and arsenoribofuranosides (Fig. 1B), found in marine algae (110), diatoms, the terrestrial alga Nostoc commune var. flagelliforme (148), and in oyster tissue (244). These structural types are not present in bacteria and fungi. However, methylarsonate and dimethylarsinate were found in several edible mushrooms (collected in Switzerland and Slovenia) and, as well, arsenobetaine (35). These materials occur in a wide range of terrestrial mushrooms and lichens; other compounds include arsenocholine and the tetramethylarsonium ion (140, 145, 146, 150, 220) and small amounts of some arsenoribofuranosides (140). The use of bioalkylation in this context appears incongruous; all of the materials contain methyl groups attached to the arsenic atom so presumably the biosynthetic process involved is biomethylation.

View larger version (10K): [in this window] [in a new window]   FIG. 1. Examples of arsenolipids. (A) O-Phosphatidyltrimethylarsonium lactic acid; (B) an arsenoribofuranoside. Several variations on this structure are observed. In each case, R is a fatty acyl residue.

The selected topics discussed here are a small component of a very wide area, the overall global role of metalloid elements. In particular, arsenic compounds are widely distributed and transformed on earth not only in the lithosphere but also in the atmosphere and hydrosphere (91, 168). Determination of arsenic species in the environment and the health effects of arsenic exposure have been reviewed (31, 49, 56, 75, 156, 164, 184, 185, 216, 235). The separation and identification of volatile arsines in gases (e.g., in headspaces above microbial cultures) is relatively straightforward, but the identification of nonvolatile components is more difficult. The nonvolatile metabolites may be converted to volatile arsines by hydride generation, for example, by reaction with sodium borohydride as a reducing agent. The advantages of hydride generation atomic absorption spectrometry (AAS) in analysis come from its much lower detection limits—parts per billion compared to parts per million for normal AAS—and from a lowered requirement upon matching the sample matrix which befalls normal AAS (59). At a pH of <4, arsenite is reduced to arsine itself, AsH₃; other conversions of oxyacids can be represented as follows: (CH₃)_nAsO(OH)_3-n (CH₃)_nAsH_3-n where n = 1 to 3 (68, 69). Hence, methylarsonate and dimethylarsinate yield, respectively, monomethylarsine and dimethylarsine. Trimethylarsine oxide yields trimethylarsine. With such techniques, nonvolatile arsenic compounds can be partially identified as mono-, di-, or trimethyl species. Hydride generation can be coupled to separation instruments such as gas chromatography-mass spectrometry (MS) but most often is used with gas chromatography using atomic absorption spectrometric detection (9). With that said, high-pressure liquid chromatography-inductively coupled plasma-MS (HPLC-ICP-MS) is probably the most powerful tool.

Also becoming more widely used for nonvolatile organometalloid analysis are even more powerful so-called hyphenated instrumental techniques such as ion or HPLC-ICP-MS (110, 141, 147). And finally, the extraction of biological materials and subsequent determination of organoarsenicals has very recently been undertaken using microwave digestion followed by UV irradiation, hydride generation, and fluorescence spectrometry (244). This is similar to microwave-assisted extraction of arsenic in soils (114).

There is a wide difference in the arsenic level of marine and terrestrial organisms. In land creatures, the arsenic level is typically about 1 ppm (dry weight), but for marine organisms the values range from a few parts per million to as much as 100 ppm (5, 149, 164). There are practical consequences. While for the U.S. population the normal adult value for urinary arsenic excretion is <50 µg day^-1, the value for Japanese adults is three times as high (179). This level of urinary arsenic probably reflects a greater dietary use of marine organisms. A detailed evaluation of "arsenic eaters" has discussed the possibility of the chronic ingestion of arsenic trioxide by otherwise healthy people in hopes of improving a range of factors, from the beauty of women, prophylaxis against disease, to increased sexual potency (197).

Early history. Hofmeister observed that subcutaneous administration of 0.06 g of sodium tellurate to a 3-kg dog gave a strong garlic odor in the expired breath of the dog 30 min later (123). Similar odors were obtained when various organ pulps were treated with tellurium compounds. Influenced by the previously described work of His on pyridine methylation, Hofmeister obtained inadequate evidence that the odorous material in the expired dog breath was "tellurmethyl," i.e., dimethyl telluride, (CH₃)₂Te. Nevertheless, he has been credited as the first to observe the biomethylation of a metalloid. Moreover, he certainly recognized the overall significance of methyl group transfer and stated that when pyridine or tellurium are administered, methylation of these compounds may occur, while under normal conditions other methylated compounds such as choline and creatine may be formed. However, he did not propose a specific methyl group donor.

Hofmeister was not the first to have noted odor production during metalloid metabolism. The formation of odorous materials from both selenium and tellurium compounds was described in animal experiments before his work; thus, a garlic smell ("Knoblauchgeruch") was observed in 1824 on dissection of a rabbit poisoned with telluric acid (99). These odors, however, were attributed to hydrogen selenide or hydrogen telluride (for details, see reference 48). Interestingly, a 1951 organic chemistry text states that dimethyl telluride has "the most abominable odour of all organometallics" (50).

ARSENIC FUNGI

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In the 17th century, a dissertation by Caroli de la Font concerning "The Nature and Causes of the Plague... deducing the Pestilential venom from the Air infected and corrupted chiefly by Arsenical Exhalations" had been reviewed in the Philosophical Transactions of the Royal Society (10). In the 1809 abridgment of this series, the dissertation was omitted and replaced with the statement that this work was "filled with absurd conjectures and reasoning" (127). However, some two centuries later, a type of toxic "arsenical exhalation" did come to prominence. Beginning about 1800, cases of poisoning in Germany were ascribed to wallpapers (and tapestries) printed with arsenical pigments.

Some of the decorative pigments used in printing were bright green arsenical materials such as Scheele's green (copper arsenite, CuHAsO₃) and Schweinfurt (or Schweinfurth) green, also termed Paris green, Vienna green, or emerald green (copper acetoarsenite, 3CuO · As₂O₃ · Cu[OOC · CH₃]). The use of these pigments was extensive (20). In 1871, arsenical papers were used "in the majority of dwelling houses, from the palace down to the navvy's hut. It is rare to meet with a house where arsenic is not visible on the walls of at least some of the rooms" (12). Moreover, arsenical pigments were used for coloring purposes in many other ways, even in foodstuffs. Many cases of poisoning were recorded (130), and a leading article in 1860 stated that "In the most emphatic manner, we feel it to be a duty to call the attention of medical practitioners to recent lamentable case of fatal poisoning, occasioned by the atrocious practice of coloring hanging-papers with arsenic pigments" (11).

The toxic effects observed in rooms coated with green pigments might have resulted from inhalation of arsenic-containing particles. However, poisoning was also observed where arsenical paper had been covered by fresh paper lacking arsenic, hence formation of dust particles was unlikely. The distinguished chemist Leopold Gmelin recorded in an 1839 newspaper article that an adverse, mouse-like odor was usually present in rooms where poisoning had occurred (100; a translation was kindly provided as a personal communication to T. G. Chasteen by M. Wiggli). He believed that the smell was caused by arsenic that had volatilized as alkarsine [cacodyl oxide, (CH₃)₂AsOAs(CH₃)₂]. Cacodyl itself is denigrated by the Oxford English Dictionary as "of most disgusting odour." Whether volatilization of arsenic was possible and caused poisoning was a much debated question (40, 157, 207, 208).

Initially, Gosio focused on arsenic volatilization by "mucor mucedo [sic]," presumably the common bread mold. Copper arsenite, the basis for the arsenical colors used in dyeing, was converted to the garlic-odored gas, but sulfide-containing pigments (e.g., orpiment and realgar) were not. Gosio aspirated gas from mixed microbial cultures into a silver nitrate solution; within 3 days there was a marked reduction of the silver salt. The presence of arsenic in the liquid after removal of silver was shown by the chemical Marsh test (102, 104). From 800 g of potato mash containing 0.12 g of arsenic trioxide, Gosio obtained 2.8 mg of metallic arsenic corresponding to the gasification of 4.3 mg of "As²O³ [sic]." This was a small yield to be sure, but it was a clear demonstration of arsenic volatilization by some saprophytes. Arsenical gases were also obtained from "hangings colored with Scheele's and Schweinfurth's greens, through the vegetation of the mucor... hence the danger to those who live in such an atmosphere." At this time, Gosio mistakenly believed that the gas contained arsine itself.

Another fungus showing a very active production of arsenical gas when grown with arsenic compounds was isolated from a piece of carrot exposed to air (102, 104). It was identical to "penicillium brevicaule [sic]" discovered earlier on rotted paper ("papier putride"). Gosio stated that this organism developed such an intensity of arsenical gas that it was dangerous to approach it ("il est dangereux de s'en approcher"). A rat exposed to the vapor was quickly killed ("Un rat qu'on expose à ces sortes d'émanations tombe rapidement dans des convulsions mortelles"), and a small mouse placed in a vessel with the fungus producing the gas died after a few seconds. When Emmerling reported contradictory results with Mucor mucedo and Aspergillus glaucus, Gosio vigorously defended his work and regretted that Emmerling had not attempted to obtain suitable cultures from him (82, 106). At the Harvard Laboratory, an American investigator, Charles Robert Sanger, had investigated cases "of chronic arsenical poisoning" from wallpapers but had been unable to demonstrate the volatilization of arsenic. Unlike Emmerling, he corresponded with Gosio, received cultures of Penicillium brevicaule, and confirmed Gosio's results (207, 208). Gosio's work was recognized by naming the garlic-odored volatile material as "Gosio gas." It is, perhaps, the only gas named eponymously.

P. brevicaule is now named formally as Scopulariopsis brevicaulis (Sacc.) Bainier (200). Historically, Penicillium and Scopulariopsis species have often been confused; there is, however, one important difference—Scopulariopsis species are never green. Scopulariopsis species, especially S. brevicaulis, are abundant in nature in material such as soil, stored grain and forage, and slowly decaying semidry vegetation (200). Quite typically, Gosio's organism was isolated from rotting paper and a slice of carrot.

Gosio had also shown volatilization of arsenic to a garlic-odored product with A. glaucus and Aspergillus virens, Mucor ramosus, Cephalothecium roseum, and Sterigmatocystis ochracea. Such organisms, termed "arsenic fungi," were later shown to be more common than previously supposed (237); thus, active strains were found among other aspergilli (Aspergillus fischeri, Aspergillus sydowi). From soil samples containing enough arsenic to prevent normal growth of certain crops, active gas producers were also obtained; they were two strains of Fusarium, a strain of Paecilomyces, and a sterile brown fungus. The volatile materials were not identified chemically. This work led to the definition of three microbial responses to arsenic: some microorganisms were inhibited by it, some tolerated arsenic but did not produce arsenical gas, and some tolerated it with production of gas. Further work with arsenic fungi is described later.

WORK OF CHALLENGER AND ASSOCIATES ("LEEDS SCHOOL")

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When Gosio gas was passed into a solution of mercuric chloride in dilute HCl (Biginelli's solution) a crystalline precipitate was formed. A small vial of the Gosio/Biginelli mercurichloride is to this day preserved at The Museum of the History of Medicine (Museo di Storia della Medicina) in Rome, Italy. Under the printed name "Laboratorio Batteriologico della Sanità Pubblica" is handwritten, "arsina penicillare comp. mercurico" (C. Serarcangeli, personal communication). From analysis of this material (25, 26) and of the gas itself (107) it appeared that the gas was diethylarsine, (C₂H₅)₂AsH. More recently, the aspiration of culture headspace gases into Biginelli's solution has been termed "chemofocusing" (65).

Trimethylarsine was obtained from the organic compounds sodium methylarsonate, CH₃AsO(ONa)₂, and sodium dimethylarsinate, (CH₃)₂AsOONa, by action of S. brevicaulis. Mixed methylarsines containing other alkyl groups were formed by growth of S. brevicaulis on suitable substrates (40) (Table 2).

View larger version (11K): [in this window] [in a new window]   FIG. 2. Typical reactions of the Challenger mechanism. The top line indicates a mechanism for the reduction, As(V) As(III), resulting in an unshared pair of electrons on As. Structures are as follows: R1 = R2 = OH, arsenate; R1 = CH3, R2 = OH, methylarsonate; R1 = R2 = CH3, dimethylarsinate. For reduction of trimethylarsine oxide to trimethylarsine, the process is a little different. Following proton addition, the structure H-O-As+(CH3)3 reacts with hydride ion leading to elimination of H2O. The bottom line indicates the methylation of an As(III) structure with SAM [shown in abbreviated form as CH3-S+-(C)2]. A proton is released and SAM is converted to S-adenosylhomocysteine [abbreviated form, S-(C)2].

View larger version (22K): [in this window] [in a new window]   FIG. 3. Challenger mechanism for the conversion of arsenate to trimethylarsine. (A) Arsenate; (B) arsenite; (C) methylarsonate; (D) methylarsonite; (E) dimethylarsinate; (F) dimethylarsinite; (G) trimethylarsine oxide; (H) trimethylarsine. The top line of structures shows the As(V) intermediates. The vertical arrows indicate the reduction reactions to the As(III) intermediates (bottom line), and the diagonal arrows indicate the methylation steps by SAM (see Fig. 2 for details of the reduction and methylation processes).

It is of interest that methylarsonous acid has been detected in human urine by using ion-pair chromatographic separation of compounds with hydride generation atomic fluorescence spectrometry detection (153, 154). The individuals who were examined normally drank water with a high arsenic level, and urine samples were analyzed after treatment of some of the subjects with the chelating agent sodium 2,3-dimercapto-1-propanesulfonate.

Understanding the chemical details of the overall scheme has sometimes caused difficulty, in part because of confusion between the term "oxidation number," which seeks to define electron distribution, and "valency" (or "valence"), which refers to the number of combining entities. The normal valency of arsenic is 3 or 5 (e.g., AsF₃ and AsF₅), and the most prominent oxidation states are -III, +III, and +V; the latter two states, As(III) and As(V), are of most concern here. The reductive steps are straightforward in concept but not in practice. Thus, arsenate with the oxidation number +V and a positive charge on the As atom is converted by a two-electron reduction to arsenite, with an oxidation number of +III [As(V) As(III)]. The other reductions are conceptually similar (Fig. 3).

To convert arsenate to trimethylarsine, the reductions are likely to be enzyme catalyzed with reductants providing the necessary 2e^-. The reductants are almost certainly thiols, and particular attention has been given to glutathione (GSH) and lipoic acid (6,8-dithiooctanoic acid). There is little experimental evidence from microbial systems, but it is assumed that the reactions resemble those for formation of methylarsonate and dimethylarsinate in mammals (239).

A disulfide redox couple, for instance involving GSH (equation 1), would drive the reduction of As(V) to As(III) in the general process of equation 2 (74).

(1)

(2)

GSH does reduce arsenate to arsenite in vitro (equation 3), and further reaction eventually forms arsenotriglutathione [tri(-glutamylcysteinylglycinyl)-trithioarsenite] (equation 4):

(3)

(4)

For the second reductive step, there is evidence that both in vitro and in vivo GSH does function as a reductant (239). Again, the mechanism is complicated by the ability of arsenic compounds to react with thiols. In model experiments, disodium methylarsonate was easily reduced by cysteine, GSH, or mercaptoethanol, to give derivatives of As(III) (74). For the overall reaction of equation 5, individual steps (equations 6, 7, and 8) were postulated:

(5)

(6)

(7)

(8)

Similar equations can be written for the subsequent reduction steps; it does seem likely that in vivo compounds with RS groups attached to the arsenic atom are involved. Dithiol derivatives such as lipoic acid could be involved, perhaps as a membrane-bound or enzyme-bound component. Thus, for arsenite itself, the reaction of equations 9 and 10 might be involved with the formation of cyclic disulfide derivatives.

(9)

(10)

The final step of the Challenger mechanism, the reduction of trimethylarsine oxide to trimethylarsine, was possible with a variety of thiol reagents (cysteine, dimercaptopropanol, lipoic acid, mercaptoacetic acid, and mercaptoethanol) (73). On the basis of kinetic and other observations, the detailed mechanism of equations 11 to 14 was postulated. Enzyme- or membrane-bound lipoic acid was regarded as a likely in vivo reductant.

(11)

(12)

(13)

(14)

If reductive steps using GSH occur in vivo, the recycling of GS-SG to GSH by GSH reductase is necessary. It is of interest that this enzyme is inhibited by various arsenic compounds, including arsenotriglutathione (230, 231).

The methylation steps are at first sight somewhat confusing, since in all cases an oxidation has occurred, As(III) As(V). To keep the process in balance there must have been a concomitant reduction. Since no redox cofactor is involved, the process appears at first glance to be a simple methyl transfer process. However, a detailed analysis of the reaction indicates that the necessary reduction is found in the corresponding decrease in oxidation number from -II to -IV for the transferred carbon. Using a formal method for assigning oxidation states to redox-labile elements in organic compounds helps in following the reduction and methylation steps with arsenic as shown in Fig. 4 (117).

View larger version (14K): [in this window] [in a new window]   FIG. 4. Changes in oxidation numbers on As and C during methylation according to Hanselmann's method (117). Abbreviations: Ox, oxidation number; E.N., electronegative (than).

Nonvolatile di- and trimethylarsenic species were identified in cultures of C. humiculus, and a dimethylarsenic compound was identified in cultures of the marine alga Polyphysa penicilus (68, 69) by the hydride generation technique (see above). When [C²H₃]methionine was used as a methyl donor, the ²H-labeled nonvolatile metabolites produced ²H-labeled arsines. These observations were consistent with the operation of the Challenger mechanism, with methyl groups being derived from methionine by way of SAM. Moreover, trimethylarsine formation from (CH₃)₂AsO(OH) by these organisms was inhibited by ethionine. Ethionine is an antagonist for methionine; hence, these observations support the role of a methionine-based synthetic path (66, 75).

ARSENIC METHYLATION

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A wood-rotting basidiomycete, Lenzites trabea, produced a garlic odor when growing on wood treated with arsenical preservatives. When 65 species of wood-rotting fungi were examined with growth in presence of As₂O₃, only L. trabea and Lenzites saepiaria produced an unidentified, garlic-odored gas (176). Another wood decaying fungus, Phaeolus schweinitzii, produced volatile methylated products of arsenic when grown with "As₂O₅" (19). The strong garlic smell aided the recognition of plates containing this organism in isolation work with a selective medium. More recent work has confirmed the volatilization of arsenic by this fungus, but again the product was not specifically identified (189). The pathogenic fungus Trichophyton rubrum produced a "peculiar, nauseating, garlic-like odor" when it was grown in the presence of arsenate or arsenite (262). The volatile product contained an unidentified arsenic component(s).

A historical connection, where the wheel comes full circle, may be noted here. The widely used wood preservative, chromated copper arsenate, is converted to trimethylarsine by the yeast C. humiculus (72). Chromated copper arsenate is quite similar to the arsenical wallpaper pigments that had triggered Gosio's work.

The work of the Leeds School was carried out by tedious chemical operations, usually the formation and analysis of mercuric chloride complexes. Moreover, the useful radioactive isotopes of arsenic were not available; however, as already noted ¹⁴C was used in studies of methyl transfer reactions. With the advent of improved analytical techniques such as gas chromatography and gas chromatography-MS, and with the availability of arsenic isotopes, the detection of trimethylarsine and related compounds can be carried out with less labor and greater certainty. A simple gas chromatographic assay was used to monitor production of trimethylarsine by three organisms isolated from sewage and described as fungi (53). Two of these organisms, G. roseum and a Penicillium species, converted methylarsonate and dimethylarsinate to trimethylarsine under a range of culture conditions; however, neither arsenate nor arsenite was volatilized. The third organism, identified tentatively as Candida humicola was said later (69) to have been reclassified as Apiotrichum humicola. It had been deposited with the American Type Culture Collection as ATCC 26699 and in the American Type Culture Collection catalog it is now described as Cryptococcus humiculus (Daszewska) Golubev. The name C. humiculus is used throughout this article; the organism is clearly a yeast. When grown at pH 5.0, it produced small amounts of trimethylarsine from arsenate, arsenite, and methylarsonate, with dimethylarsinate being the best substrate for volatilization.

The variables affecting the methylation of arsenic by C. humiculus have been studied. Only a brief account will be given here since two authoritative reviews are available (52, 75). All of the organic As(V) compounds of the Challenger pathway (methylarsonate, dimethylarsinate, trimethylarsine oxide) have been observed in cell extracts metabolizing arsenate, and all of them are precursors for trimethylarsine biosynthesis (70, 195). Preconditioning of cells with dimethylarsinate improves trimethylarsine formation from arsenate and dimethylarsinate. In later experiments, the nonvolatile components of C. humiculus and S. brevicaulis were identified (67). These experiments used low levels (1 ppm) of the arsenic substrates, and under these conditions there was no detectable formation of trimethylarsine with either organism.

With methylarsonate as substrate, C. humiculus produced low levels of dimethylarsinate and trimethylarsine oxide by the second day, with slow increases of both metabolites over 4 weeks. For S. brevicaulis, the metabolism was much slower, with trace amounts of dimethylarsinate and trimethylarsine oxide not appearing until after 2 weeks of incubation.

This was the first report of trimethylarsine oxide as an end product metabolite for the two organisms; under the experimental conditions, it was the major product rather than trimethylarsine. Apparently the organisms tolerated low levels of this oxide, and further "detoxification" by conversion to trimethylarsine was not necessary. An extended model (Fig. 5) for arsenic methylation involving both cells and medium was proposed (67). The observed reaction rates suggested a possible sequential transfer of 2 methyl groups from SAM to methylarsonate as indicated. These observations emphasized that the methylation of arsenic by microorganisms is a very complex process despite the general simplicity of the Challenger mechanism.

View larger version (15K): [in this window] [in a new window]   FIG. 5. Expanded version of the Challenger mechanism. This mechanism, proposed for C. humiculus, indicates roles for components both in the cells themselves and in the culture medium. The double vertical lines indicate cell walls. (A) Phosphate transport system; (B) thiols and/or dithiols; (C) active transport system; (D) active/passive transport; (E) passive diffusion. Abbreviations: MMAV, methylarsonic acid; DMA, dimethylarsinic acid; TMAO, trimethylarsine oxide. This diagram is redrawn from scheme 2 of reference 67.

C. humiculus produced a garlic odor when grown in the presence of benzenearsonic acid (phenylarsonic acid, C₆H₅As[OH]₂) (65). By use of the chemofocusing technique, the volatile product was shown to be C₆H₅As(CH₃)₂. Similarly, methylphenylarsinic acid, (C₆H₅)(CH₃)AsO(OH), was converted to the same dimethylphenylarsine. Arsanilic acid, H₂NC₆H₄AsO(OH)₂, was not methylated, but 2-hydroxy-4-aminobenzene-arsonic acid yielded trimethylarsine.

The fungicide methylarsine sulfide, (CH₃AsS)_x (Rhizoctol), produced a garlic odor when grown with C. humiculus; the volatile material was a mixture of trimethylarsine and methylarsine in the ratio 90:10. The conversion was rapid, with 50% of added arsenic being converted in 3 days; compare arsenate or dimethylarsinate trimethylarsine, which resulted in 1% conversion in 10 days (71). Methylarsine sulfide was actually more toxic to C. humiculus than either arsenite or the corresponding oxide, ([CH₃]₂AsO)_x, suggesting that at least in this case, methylation was not a detoxification process. Methylarsine oxide was also rapidly transformed by C. humiculus to dimethylarsinate (24 h); small amounts of unidentified volatile arsines were also formed.

The marine yeast Rhodotorula rubra an obligate aerobe, converted arsenate to arsenite, methylarsonate, and dimethylarsinate. Unidentified "volatile arsines" were also produced (243).

Modern analytical techniques for the determination of arsenic species continue to be developed and used (64, 110, 147, 149, 151-154, 190-192, 244). In one study a Penicillium species was isolated from an agricultural evaporation pond in an area with high concentrations of soil arsenic. The organism formed trimethylarsine from methylarsonate; addition of carbohydrate and sugar acids to the minimal medium suppressed volatilization, whereas glutamine, isoleucine, and phenylalanine had a stimulatory effect (128). Various elements known to be toxic to living systems generally inhibited the conversion of methylarsonate to trimethylarsine by this Penicillium sp. (92). The most inhibitory element was V, followed by Ni, Sn, B, Te, Mn, Mo, Cr, and Ag; at low concentrations the following elements were stimulatory: Hg, Fe, Cu, Al, Zn, and Se. In soil contaminated more than 30 years ago by arsenic trioxide from a cattle dipping vat, a low level of in situ volatilization of arsenic was demonstrated by subsurface probes. From this soil, a Fusarium sp. producing unidentified volatile arsenicals was isolated (238). A recent monograph provides more information in this area (92a).

The metabolism of arsenic compounds widely used in agriculture has been studied in mixed microbial populations from soil. While evidence was obtained for arsenic volatilization, it was usually not clear whether bacteria or fungi were involved. For example, when ¹⁴C-labeled dimethylarsinate was added to three soil samples under aerobic conditions, 35% of the added arsenic was converted to a volatile material or materials over a 24-week period, and 41% was converted to ¹⁴CO₂ and AsO₄^3- (253). Under conditions considered to be anaerobic, the amount of volatilized arsenic increased to 61% and a garlic-like odor was observed. If the conditions (flooding with water) were indeed anaerobic, fungal growth would have been unlikely, and the observed volatilization might have resulted from a mixed bacterial culture. The volatilization of arsenic from a soil treated with [⁷⁴As]H₃AsO₄, sodium [¹⁴C]methylarsonate, and [¹⁴C]dimethylarsinate was most rapid under aerobic conditions, but significant yields of di- and trimethylarsine were obtained when the experimental flasks were swept with nitrogen (252). The formation of methylarsine was not detected.

Air samples collected over sodium arsenite-treated lawn grass for 2-day periods, with sample collections usually at 2- or 3-h intervals, reliably gave small amounts of trimethylarsine after about 20 h (29). Dimethylarsine was observed in small amounts only in the last two 2-h intervals. From methylarsonate, trimethylarsine formation began after the first 2-h interval and continued throughout the experiment with substantial amounts after about 14 h. Specific organisms were not isolated from the mixed cultures, but the formation of dimethylarsine, not usually a fungal product, suggests bacterial action.

When three soil samples were incubated with arsenate, arsenite, methylarsonate, and dimethylarsinate, methyl- and dimethylarsine were only produced from methylarsonate and dimethylarsinate; some arsine production was also observed. Surprisingly, two soil bacteria (a Pseudomonas sp. and an Alcaligenes sp.) produced only arsine when incubated anaerobically with arsenate or arsenite (51). Arsine (AsH₃) formation is relatively uncommon but is discussed later in connection with anaerobic organisms.

Five bacterial species, (Corynebacterium sp., E. coli, Flavobacterium sp., Proteus sp., and Pseudomonas sp.) isolated from the environment and acclimatized to growth with sodium arsenate for 6 months, transformed arsenate to arsenite, and all of them produced dimethylarsine. The Pseudomonas sp. formed all three of the methylated arsines (214). Arsenic accumulated by the cells was in a protein fraction (212). Six bacterial species (Achromobacter sp., Aeromonas sp., Alcaligenes sp., Flavobacterium sp., Nocardia sp., and Pseudomonas sp.) produced both mono- and dimethylarsine from methylarsonate; only two of them produced trimethylarsine. A Nocardia sp. was the only organism that produced all of the methylarsines from this substrate (213).

C. humiculus was known to reduce trimethylarsine oxide to trimethylarsine (195), and this oxide was also reduced under both aerobic and anaerobic conditions by several bacteria (194). The most active (142 nmol min^-1 g of cells^-1 [wet weight] at 37°C) of four anaerobes (Fusobacterium nucleatum, Veillonella alcalescens, and two unidentified skin organisms) was V. alcalescens. However, anaerobically grown Staphylococcus aureus gave the highest rate for any of the organisms tested anaerobically (208 nmol min^-1 g of cells^-1 [wet weight]). Similar results were obtained using aerobic organisms; a marine pseudomonad showed the highest activity (585 nmol min^-1 g of cells^-1 [wet weight]) with substantial activity (range, 80 to 170 nmol min^-1 g of cells^-1 [wet weight]) for B. subtilis, E. coli, S. aureus (grown aerobically), and two unidentified skin aerobes. A further skin aerobe and Streptococcus sanguis were much less active. The same conversion was shown by river water, some sea sediments and sewage sludge (low levels in both cases analyzed by odor alone), and rumen fluid (under both aerobic and anaerobic conditions). The authors noted that "the odor of trimethylarsine becomes immediately apparent if some of the oxide is placed on the skin," presumably by the action of the mixed microbial flora (194).

Arsenic-resistant Pseudomonas putida, isolated from a contaminated Chlorella culture, contained a nonvolatile trimethylarsenic species and excreted into the medium all three methylated species (170). Similar results were obtained with arsenic-resistant Klebsiella oxytoca and a Xanthomonas sp. (169). None of these organisms formed volatile arsenic compounds.

A sample of soil with a low level of arsenic contamination (As, 1.5 ppm) yielded two arsenic-resistant, nonmethylating bacteria (Bacillus sp. and Pseudomonas fluorescens) and a new, arsenic-resistant organism, assigned to the Flavobacterium-Cytophaga groups, with methylating capacity. It produced only trimethylarsine as the volatile material; arsenite was transformed more rapidly than arsenate. Dimethylarsinate did not yield trimethylarsine (124). Contaminated soil (contaminated with As, Cr, and Cu) from a wood-impregnating plant had methylating activity (production of methylarsonate and dimethylarsinate). There was no mention of arsenic volatilization in this work (241).

A brief report has described the bioleaching of columns of arsenic-contaminated soil percolated with a nutrient medium (240). Arsenic was converted to unidentified volatiles, most efficiently under anaerobic conditions, thus suggesting possible bacterial action. Using garden soil spiked with As₂S₃, the undefined microbial population converted the insoluble material to "nearly 50% water-soluble form within two months" (240). At the same time, small amounts of unidentified volatile arsenicals were produced. Bacterial leaching of arsenic-contaminated materials was suggested as a possible means for bioremediation. Solubilization of arsenic by oxidation from the mineral arsenopyrite (FeAsS) by mesophilic and moderately thermophilic acidophiles has been described without mention of any formation of volatile products (79, 240).

Further work on the anaerobic volatilization of arsenic has focused on anaerobic ecosystems such as sewage sludge and the use of pure cultures of anaerobes. As noted earlier, sewage sludge had yielded arsenic volatilizing organisms in 1973. Soon thereafter, sludge from an anaerobic digester was treated with [⁷⁴As]Na₂HAsO₄ and then incubated anaerobically (172, 173). Volatile, radioactive arsenic compounds were produced, as evidenced by the use of rubber traps, but when the sludge was preheated to 90°C for 15 min, volatilization did not occur. Unfortunately, experimental difficulties with the system precluded extensive observations. Some evidence for the assumption that methanogens were producing dimethylarsine was obtained by taking advantage of the fact that the main precursor of methane formation in sewage sludge was carbon atom 2 (the methyl carbon) of acetate. In experiments with [2-¹⁴C]acetate, radioactivity was incorporated into the traps only when arsenate was present. Other preliminary experiments indicated that rumen fluid and compost were particularly effective in volatilizing arsenic from [⁷⁴As]Na₂HAsO₄.

A more-sophisticated analytical technique (on-line coupling of gas chromatography with ICP-MS) later detected volatile metal and metalloid species in landfill and sewage gases (85, 86, 121). For arsenic, the gases from both sources contained (CH₃)₂AsH, (CH₃)₃As, and, interestingly, (CH₃)₂C₂H₅As. Only sewage gas formed CH₃AsH₂, and only landfill gas formed (C₂H₅)₃As. Particularly noteworthy is the formation of the ethylated species. Assuming that the ethyl group transfers are a result of microbial action and thus an instance of bioalkylation, it would be of considerable interest to identify the organisms responsible. Similar work has shown that gases released from anaerobic wastewater treatment plants contained arsine as well as the three trimethylarsines (178).

An interesting feature of the work with the anaerobic ecosystems just described was the formation of arsine itself (86, 178). Although the formation of arsine from arsenate and arsenite by soil bacteria (Pseudomonas sp. and Alcaligenes sp.) was known (51), the microbial formation of this nonmethylated metabolite is uncommon. However, as will be seen, arsine formation by pure cultures of anaerobes has been described.

Cell extracts of Desulfovibrio vulgaris strain 803 produced an unidentified, garlic-odored gas when incubated with sodium arsenate (175), and unpublished results (75) have indicated the production of both di- and trimethylarsine from arsenate by cell extracts of Methanobacterium thermoautotrophicum. When this organism was grown under stable chemostat conditions with H₂ and CO₂ as growth substrates, arsenate was transformed to volatile products, mainly arsine itself with small amounts of dimethylarsine (17). Lowered phosphate concentrations increased the efficiency of the volatilization by 25%.

Enrichment cultures of anaerobes isolated from an arsenic-contaminated lake in the Canadian sub-Arctic region with five selective media showed extensive formation of methylarsenicals (30). Anaerobic oligotrophs were most active, producing a high proportion of dimethylarsinate. Sulfate-reducing consortia with acetate produced mainly trimethylarsine oxide, with relative concentrations of methylarsonate and dimethylarsinate increasing with time. Sulfate-reducing organisms with lactate produced only trimethylarsine oxide by day 4, with a range of products being produced on extended incubation. Iron- and manganese-reducing organisms grew poorly, producing only low levels of methylated arsenicals. Some of the compounds were probably methylarsenic(III) thiols, and volatile arsines were also produced in some cases. In sulfate-reducing (acetate) cultures, AsH₃ was formed with monomethylarsine as the major volatile compound. Sulfate-reducing (lactate) cultures gave AsH₃ and trimethylarsine as the major products.

Several representatives of sewage sludge microflora (methanogenic archaea) and sulfate-reducing anaerobes have been examined in pure culture in the presence of various arsenic concentrations (178). The most efficient organism for arsenic volatilization was Methanobacterium formicicum; arsine and all three methylarsines, as well as an unidentified volatile arsenic compound, were produced over the As concentration range of 0.05 to 0.3 mM KH₂AsO₄. Methanosarcina barkeri produced only arsine over the same range, and Methanobacterium thermoautotrophicum formed arsine at 0.1, 0.3, and 0.5 mM concentrations, especially so at the higher arsenic levels. Three organisms—Clostridium collagenovorans, D. vulgaris, and Desulfovibrio gigas—produced small amounts of trimethylarsine. The facultative marine anaerobe Serratia marinorubra converted arsenate to arsenite and methylarsonate when grown aerobically; volatile arsines were not produced under either aerobic or anaerobic conditions (243).

Since arsenite inhibited methane biosynthesis, it seemed possible that methyl transfer to arsenite led to the formation of a nonvolatile compound. By the use of [¹⁴CH₃]methylcobalamin, this compound was identified as methylarsonate (175). An abbreviated, Challenger-like mechanism was postulated for dimethylarsine synthesis, with dimethylarsinate undergoing a 4e^- reduction as the final step: arsenate arsenite methylarsonate dimethylarsinate dimethylarsine. Unfortunately, in the schematic mechanism presented by these authors, there appears to be confusion between oxidation numbers and valency. The methyl groups were represented as derived from methylcobalamin with formation of vitamin B_12r (notation representing the demethylated form of B₁₂).

One difficulty is the existence of nonenzymatic methyl transfers from this methylcobalamin, e.g., to mercury (251). The methylation of arsenite by methylcobalamin occurred at a low rate in the presence or absence of rat liver extract (33). In 1973, the nonenzymatic transfer of the methyl group of methylcobalamin to arsenite was shown to require a reducing agent such as dithioerythritol (209, 210). Typically, incubation of methylcobalamin, As₂O₃, and dithioerythritol in water gave arsine, methylarsine, and methane. The reductant, Zn/NH₄Cl, behaved similarly. Using [C²H₃]cobalamin, the arsenical product was C²H₃AsH₂, demonstrating transfer of an intact methyl group. There was no alkyl transfer from ethylcobalamin. Selenite inhibited arsenite methylation and selenite underwent methylation to dimethylselenide in the absence of arsenite.

Arsenite methylation by methylcobalamin in the presence of GSH has also been observed (259). The reaction was studied in capped tubes, one reason for this being "to prevent any as yet unknown volatile intermediates from escaping" (259). After treatment of the reaction mixtures with H₂O₂ to oxidize arsenical intermediates to the As(V) state, methylarsonate and smaller amounts of dimethylarsinate were identified. In view of this experimental procedure, the initial products of methyl transfer were not identified. In addition to containing arsenite, methylcobalamin, and GSH, the complex incubation mixtures contained vitamin B₁₂ and SAM. The unexplained presence of the latter slightly increased the yield of methylarsonate; in the absence of GSH, methylation was not observed. Sodium 2,3-dimercapto-1-propane-sulfonate and sodium selenite also functioned as reducing agents; in combination these two agents behaved synergistically.

Work with E. coli cultures isolated from rats fed dimethylarsinate for 6 months has shown that cysteine but not GSH was required for dimethylarsinate metabolism. Thus, although GSH contributes to arsenic metabolism in animals, there may be a different metabolic pathway in bacteria (147).

Some workers add vitamin B₁₂ (34) to assays for methyl transfer in animal extracts, but there is little evidence for the requirement. Methylcobalamin, added to incubations of rat liver cytosol, produced no effect (228). However, for arsenic methylation by the microbial contents of mouse intestinal cecum, methylcobalamin addition increased production of methylarsonate from inorganic forms of arsenic but had little effect on the formation of dimethylarsinate. These increases may have resulted from increased production of SAM by the cecal microflora or from the use of methylcobalamin in undefined enzymatically catalyzed methylation reactions (116).

The possible chemistry for nonenzymatic and enzyme-catalyzed methyl transfers from methylcobalamin to arsenic has been discussed in detail (83, 196, 203, 209, 210, 250). There are strongly held opinions, particularly with respect to the methyl donor situation in anaerobic organisms. Wood et al. stated that, taking into account all of the available experimental data, "we believe that the biomethylation of arsenic by methyl-B₁₂ occurs in anaerobic ecosystems" (203). However, Cullen and Reimer claimed that "until strong evidence is advanced, there seems little need to invoke a different mechanism for arsenic biomethylation by bacteria from that discussed above for fungi" (75). Moreover, in a discussion at a 1978 symposium dedicated to Challenger, Cullen expressed himself more forcefully: "We are brain-washed by the idea that methyl B₁₂ is involved in all of these reactions. We firmly believe that there is no B₁₂ involved in arsenic methylation. Professor Wood pointed out that the nature of the methylating species had to be established in nature. Much of our discussion now involves base-on, base-off details, yet the compound may not in fact be the methylating agent" (62). It should be noted that the literature contains confusing statements with respect to oxidation numbers and electron flow details (66, 261).

Clearly, there is no general consensus as to a mechanism for anaerobic dimethylarsine formation. There are two general explanations for the observed results with M. bryantii: either there is a direct transfer from methylcobalamin or, if not, then methylcobalamin transfers methyl to a further carrier molecule. Three mechanisms are available for the direct transfer of methyl from methylcobalamin, represented as CH₃-Co(III). The methyl group may be transferred as an anion (equation 15), as a radical (equation 16), or as a cation (equation 17):

(15)

(16)

(17)

In terms of the enzymatic situation, the cation transfer (equation 17) would appear to be advantageous; it is analogous to the situation with SAM. This mechanism requires formation of ^-Co(I), also represented as B_12s. However, the compound formed after methyl transfer was B_12r (equation 16); it was described as the normal cobamide product formed by demethylation of methylcobalamin (175). If correct, this identification suggests the intervention of a methyl radical, and in fact, consideration of the standard reduction potential (-0.67 V) for As(V) or As(III) is suggestive of a reductive homolytic cleavage (83, 249).

An alternative explanation (209, 210) invoked a role for a thiol, likely GSH, in formation of methane, acetate, and methylarsines by anaerobes. For the first two metabolites, the process is more complex than originally postulated, but the overall concept may be valid for methyl transfer to arsenic. The role of the thiol is that of a reductant; in fact, methylcobalamin can be reductively cleaved nonenzymatically in neutral or weakly acid solutions to form methane. The addition of the thiol to the cobalt atom promoted the heterolytic cleavage of the Co-C bond, leading to formation of a methyl carbanion (or similar species) which may be enzyme bound. Such a species was described as a "kryptocarbanion"; however, "cryptocarbanion" would be more appropriate.

For the enzyme reaction, two thiol units were involved as well as linkage of methylcobalamin as a corrinoid protein (Fig. 6). The methyl carbanion was represented as attacking arsenic in the ionized state and carrying three positive charges (equations 18 to 20). For arsenite the ionization would be represented as As(OH)₃ As³⁺(OH^-)₃. Other species would presumably be possible, for instance, those with GSH replacing the OH groups: As(SG)₃ As³⁺(SG^-)₃.

(18)

(19)

(20)

The ionized species, CH₃As²⁺, would correspond to methylarsonate or a similar structure [CH₃As(OH)₂ CH₃As²⁺(OH^-)₂], and (CH₃)₂As⁺ would correspond to dimethylarsinate [(CH₃)₂AsOH (CH₃)₂As⁺(OH^-)] in the usual Challenger mechanism. This proposed mechanism requires production of Co(I), i.e., B_12s rather than the observed B_12r. Perhaps an interconversion occurred during workup of the enzymatic incubation mixtures. For the nonenzymatic transfer, this mechanism was dismissed (259) as "less likely in the highly reductive conditions of our experiments," and instead a "nucleophilic attack on the Co-C bond by an arsenite-GSH complex" was proposed. It is not clear why the reaction conditions for this work were characterized as "highly reducing." The tubes, although capped, contained air, and the incubation components in addition to arsenite and methylcobalamin were the required GSH in Tris-HCl buffer with Mg²⁺, SAM and vitamin B₁₂. "Highly reducing" seems to be an exaggeration.

View larger version (6K): [in this window] [in a new window]   FIG. 6. Possible mechanism for formation of a "cryptocarbanion" from a methyl cobalamin. E indicates an enzyme containing two SH groups (209, 210).