Production of toxic volatile trimethylbismuth by the intestinal microbiota of mice.

The biotransformation of metals and metalloids into their volatile methylated derivatives by microbes growing under anaerobic conditions (e.g., the mammalian intestinal microbiota) plays an important role in spreading these compounds in the environment. In this paper, we could show that the presence of an intact intestinal microbiota of mice provides the conditio sine qua non for the production of these mostly toxic derivatives. To document the indispensible role of the intestinal microbiota in methylating metals and metalloids to volatile derivatives under in vivo conditions, we compared the methylation capability of conventionally raised (CONV) and germ-free (GF) B6-mice fed with chow containing colloidal bismuth subcitrate (CBS) as the starting material for the formation of volatile methylated metal(loid)s. Permethylated volatile trimethylbismuth ((CH(3))(3)Bi) was only detected in the blood of the conventionally raised mice. Concomitantly, a higher bismuth concentration was found in organs such as liver, lung, testicles, and brain of the CONV mice as compared to those of GF mice (P > 0.01), strongly suggesting a correlation between the intestinal biomethylation of bismuth and its accumulation in mammalian tissues.

Connection between multimetal(loid) methylation in methanoarchaea and central intermediates of methanogenesis.

In spite of the significant impact of biomethylation on the mobility and toxicity of metals and metalloids in the environment, little is known about the biological formation of these methylated metal(loid) compounds. While element-specific methyltransferases have been isolated for arsenic, the striking versatility of methanoarchaea to methylate numerous metal(loid)s, including rare elements like bismuth, is still not understood. Here, we demonstrate that the same metal(loid)s (arsenic, selenium, antimony, tellurium, and bismuth) that are methylated by Methanosarcina mazei in vivo are also methylated by in vitro assays with purified recombinant MtaA, a methyltransferase catalyzing the methyl transfer from methylcobalamin [CH3Cob(III)] to 2-mercaptoethanesulfonic acid (CoM) in methylotrophic methanogenesis. Detailed studies revealed that cob(I)alamin [Cob(I)], formed by MtaA-catalyzed demethylation of CH3Cob(III), is the causative agent for the multimetal(loid) methylation observed. Moreover, Cob(I) is also capable of metal(loid) hydride generation. Global transcriptome profiling of M. mazei cultures exposed to bismuth did not reveal induced methyltransferase systems but upregulated regeneration of methanogenic cofactors in the presence of bismuth. Thus, we conclude that the multimetal(loid) methylation in vivo is attributed to side reactions of CH3Cob(III) with reduced cofactors formed in methanogenesis. The close connection between metal(loid) methylation and methanogenesis explains the general capability of methanoarchaea to methylate metal(loid)s.

Toxicity of Methylated Bismuth Compounds Produced by Intestinal Microorganisms to Bacteroides thetaiotaomicron, a Member of the Physiological Intestinal Microbiota.

Methanoarchaea have an outstanding capability to methylate numerous metal(loid)s therefore producing toxic and highly mobile derivatives. Here, we report that the production of methylated bismuth species by the methanoarchaeum Methanobrevibacter smithii, a common member of the human intestine, impairs the growth of members of the beneficial intestinal microbiota at low concentrations. The bacterium Bacteroides thetaiotaomicron, which is of great importance for the welfare of the host due to its versatile digestive abilities and its protective function for the intestine, is highly sensitive against methylated, but not against inorganic, bismuth species. The level of methylated bismuth species produced by the methanoarchaeum M. smithii in a coculture experiment causes a reduction of the maximum cell density of B. thetaiotaomicron. This observation suggests that the production of methylated organometal(loid) species in the human intestine, caused by the activity of methanoarchaea, may affect the health of the host. The impact of the species to reduce the number of the physiological intestinal microbiota brings an additional focus on the potentially harmful role of methanoarchaea in the intestine of a higher organism.

Investigation of biomethylation of arsenic and tellurium during composting.

Though the process of composting features a high microbiological activity, its potential to methylate metals and metalloids has been little investigated so far in spite of the high impact of this process on metal(loid) toxicity and mobility. Here, we studied the biotransformation of arsenic, tellurium, antimony, tin and germanium during composting. Time resolved investigation revealed a highly dynamic process during self-heated composting with markedly differing time patterns for arsenic and tellurium species. Extraordinary high concentrations of up to 150 mg kg(-1) methylated arsenic species as well as conversion rates up to 50% for arsenic and 5% for tellurium were observed. In contrast, little to no conversion was observed for antimony, tin and germanium. In addition to experiments with metal(loid) salts, composting of arsenic hyperaccumulating ferns Pteris vittata and P. cretica grown on As-amended soils was studied. Arsenic accumulated in the fronds was efficiently methylated resulting in up to 8 mg kg(-1) methylated arsenic species. Overall, these studies indicate that metal(loid)s can undergo intensive biomethylation during composting. Due to the high mobility of methylated species this process needs to be considered in organic waste treatment of metal(loid) contaminated waste materials.

Organoarsenicals. Uptake, metabolism, and toxicity.

Arsenic is categorized by the WHO as the most significant environmental contaminant of drinking water due to the prevalence of geogenic contamination of groundwaters. Arsenic and the compounds which it forms are considered to be carcinogenic. The mechanism of toxicity and in particular of carcinogenicity of arsenic is still not well understood. The complexity originates from the fact that arsenic can form a rich variety of species, which show a wide variability in their toxicological behavior. The process of biomethylation was for many years regarded as a detoxification process; however, more recent research has indicated that the reverse is in fact the case. In this book chapter we give a summary of the current state of knowledge on the toxicities and toxicological mechanisms of organoarsenic species in order to evaluate the role and significance of these regarding their adverse effects on human health.

Biovolatilization of metal(loid)s by intestinal microorganisms in the simulator of the human intestinal microbial ecosystem.

Methylation and hydrogenation of metal(loid)s by microorganisms are widespread and well-known processes in the environment by which mobility and in most cases toxicity are significantly enhanced in comparison to inorganic species. The human gut contains highly diverse and active microbiocenosis, yet little is known about the occurrence and importance of microbial metal(loid) methylation and hydrogenation. In this study, an in vitro gastrointestinal model, the Simulator of the Human Intestinal Microbial Ecosystem (SHIME),was used for investigating volatilization of metal(loid)s by intestinal microbiota. Suspensions from different compartments of the SHIME system analogous to different parts of the human intestinal tract were incubated with different concentrations of inorganic Ge, As, Se, Sn, Sb, Te, Hg, Pb, and Bi and analyzed by gas chromatography and inductively coupled plasma mass spectrometry (GC-ICP-MS). Significant volatilization was found for Se, As, and Te (maximal hourly production rates relative to the amount spiked; 0.6, 2, and 9 ng/mg/h, respectively). In addition, volatile species of Sb and Bi were detected. The occurrence of AsH3 and (CH3)2Te was toxicologically important. Furthermore, mixed Se/S and mixed As/S metabolites were detected in significant amounts in the gas phase of the incubation experiments of which two metabolites, (CH3)2AsSSCH3 and CH3As(SCH3)2, are described for the first time in environmental matrices. The toxicology of these species is unknown. These data show that the intestinal microbiota may increase the mobility of metal(loid)s, suggesting a significant modulation of their toxicity. Our research warrants further studies to investigate the extent of this process as well as the availability of metal(loid)s from different sources for microbial transformations.

Determination of 13C/12C isotopic ratios of biogenic organometal(loid) compounds in complex matrixes.

Methylated metal(loid) compounds are formed in the environment by abiotic as well as enzymatically catalyzed transfer of a methyl group. Due to the increased mobility and toxicity in comparison to the inorganic precursors, the investigation of the formation process is of high relevance. Though the natural abundance carbon isotope ratio can give important insights toward their origin as well as the biochemical methyl transfer process, so far, these species have not been investigated by carbon isotope ratio mass spectrometry (IRMS). This is due to the analytical challenge to precisely determine the natural isotope distribution of trace amounts of metal(loid)-bound carbon in complex organic matrixes. To overcome this problem, we tested the concept of selective derivatization of nonvolatile organometal(loid)s by hydride generation (HG) followed by purge and trap (P-T) enrichment, heart-cut gas chromatography (hcGC), and subsequent analysis by GC/IRMS. Parameter optimization of HG/P-T/hcGC was conducted using online coupling to element-sensitive ICPMS (inductively coupled plasma mass spectrometry) detection. The purity of the HG/P-T/hcGC fraction was verified by GC/MS. For the model substance trimethylarsine oxide (TMAsO), an excellent agreement of the delta(13)C-value analyzed by HG/P-T/hcGC-GC/IRMS was achieved in comparison to the bulk delta(13)C-value, which shows that no significant isotope fractionation occurred during hydride generation and subsequent separation. The optimized method showed good reproducibility and a satisfying absolute detection limit of 4.5 microg TMAsO (1.2 microg(carbon)). This method was applied to the analysis of TMAsO in compost. The low delta(13)C value of this compound (-48.38 +/- 0.41 per thousand) indicates that biomethylation leads to significant carbon fractionation. HG/P-T/hcGC-GC/IRMS is a promising tool for investigation of the biomethylation process in the environment.

Methylated arsenic, antimony and tin species in soils.

Methylated species of antimony, arsenic and tin were examined in urban soils of the Ruhr basin, near the cities of Duisburg and Essen, Germany. The main aim of this study was to investigate the occurrence of mono-, di- and trimethylated species of these elements in urban soils. The influence of historical and present land use upon the species content was examined. The distribution of inorganic As, Sb and Sn and their methylated species along the profile depth was investigated. As, Sb and Sn speciation was performed by pH-gradient hydride generation purge and trap gas chromatography, followed by inductively-coupled plasma mass spectrometry (HG-PT-GC/ICP-MS). Species' structures were confirmed by GC-EI/MS-ICP-MS. Monomethylated Sb and As were the dominant species detected: the concentration of these metal(loid) species varied between <0.07-56 microg kg(-1) per dry mass. All dimethylated species and monomethyltin concentrations were between <0.01-7.6 microg kg(-1) per dry mass, and for the trimethylated species of all examined elements, concentrations between <0.001-0.63 microg kg(-1) per dry mass were detected. The highest organometal(loid) concentrations were observed in agricultural soils and garden soils; lower concentrations were found in the soils of abandoned industrial sites (wasteland, primary forest and grassland) and a flood plain soil of the Rhine. This result can be ascribed to both the cultivation and the increased biological activity of the agricultural soils, and the generally higher contamination, the disturbed structure and the artificial substrates (deposits from industrial sources) of the abandoned industrial soils. Due to periodical sedimentation, the flood plain profile was the only one where no depth dependence of organometal(loid) species concentration was detected. The other soil profiles showed a decrease of species content with increasing depth; this was particularly noticeable in soils with a clear change from a horizon with an organic character towards a mineral horizon, i.e. decreasing vitality from profile top to bottom. It is not as yet clear whether the organometal(loid) species are formed in the mineral horizons of the profiles or whether they are displaced from the organic, biologically-active horizons towards the mineral horizons. Field studies revealed that soil parameters like pH, water content or temperature did not correlate significantly with the degree of biomethylation observed. In contrast to the lower in vitro biomethylation efficiency of Sb vs. As in microbial incubations, we consistently detected higher proportions of transformed Sb compounds in situ in soil samples. These data may indicate a need to re-examine the currently accepted model of Sb biogeochemical cycling in the real environment.