A.W. Rettenmeier

Environmental Distribution, Analysis, and Toxicity of Organometal(loid) Compounds

ABSTRACT The biochemical modification of the metals and metalloids mercury, tin, arsenic, antimony, bismuth, selenium, and tellurium via formation of volatile metal hydrides and alkylated species (volatile and involatile) performs a fundamental role in determining the environmental processing of these elements. In most instances, the formation of such species increases the environmental mobility of the element, and can result in bioaccumulation in lipophilic environments. While inorganic forms of most of these compounds are well characterized (e.g., arsenic, mercury) and some of them exhibit low toxicity (e.g., tin, bismuth), the more lipid-soluble organometals can be highly toxic. Methylmercury poisoning (e.g., Minamata disease) and tumor development in rats after exposure to dimethylarsinic acid or tributyltin oxide are just some examples. Data on the genotoxicity (and the neurotoxicity) as well as the mechanisms of cellular action of organometal(loid) compounds are, however, scarce. Many studies have shown that the production of such organometal(loid) species is possible and likely whenever anaerobic conditions (at least on a microscale) are combined with available metal(loid)s and methyl donors in the presence of suitable organisms. Such anaerobic conditions can exist within natural environments (e.g., wetlands, pond sediments) as well as within anthropogenic environmental systems (e.g., waste disposal sites and sewage treatments plants). Some methylation can also take place under aerobic conditions. This article gives an overview about the environmental distribution of organometal(loid) compounds and the potential hazardous effects on animal and human health. Genotoxic effects in vivo and in vitro in particular are discussed.

Direct identification of bacteria in urine samples by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and relevance of defensins as interfering factors

Standard methods for the identification of uropathogens that are based on the determination of metabolic activity require cultivation on agar plates, which often takes more than 1 day. If microbial growth on agar plates is slow, or if metabolic activity is impaired by adverse interactions resulting from the patients condition or from medical treatment, the application of standard methods may lead to delayed or erroneous identification of bacteria. In recent studies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be able to rapidly identify bacteria obtained from cultures. We tested the applicability of this analytical technique for the rapid identification of bacteria collected directly from urine samples and compared the results with those of conventional identification methods, such as the Vitek system, the MicroScan WalkAway system and the API system, and in some cases with the gas chromatographic determination of the bacterial long-chain fatty acid pattern. We analysed a total of 107 urine samples with bacterial counts ranging from 10(2) to ≥10(5) c.f.u. ml(-1). Mass spectrometric identification of bacteria was accomplished for 62 of these samples. In the mass spectra obtained from 40 of the 45 urine samples for which no identification result was achieved, a triplet of very intense peaks corresponding to the human α-defensins 1, 2 and 3 occurred at m/z values of around 3440 Da. This signal suppressed the intensity of the bacterial protein peaks and thus impaired database matching. Our results show that MALDI-TOF MS allows the reliable direct identification of bacteria in urine samples at concentrations as low as 10(3) c.f.u. ml(-1). In a subset of samples, human defensins may occur and impair the mass spectrometric identification of bacteria.

Cellular uptake, subcellular distribution and toxicity of arsenic compounds in methylating and non-methylating cells ☆

Arsenic is a known human carcinogen, inducing tumors of the skin, urinary bladder, liver and lung. Inorganic arsenic, existing in highly toxic trivalent and significantly less toxic pentavalent forms, is methylated to mono- and di-methylated species mainly in the liver. Due to the low toxicity of pentavalent methylated species, methylation has been regarded as a detoxification process for many years; however, recent findings of a high toxicity of trivalent methylated species have indicated the contrary. In order to elucidate the role of speciation and methylation for the toxicity and carcinogenicity of arsenic, systematic studies were conducted comparing cellular uptake, subcellular distribution as well as toxic and genotoxic effects of organic and inorganic pentavalent and trivalent arsenic species in both non-methylating (urothelial cells and fibroblasts) and methylating cells (hepatocytes). The membrane permeability was found to be dependent upon both the arsenic species and the cell type. Uptake rates of trivalent methylated species were highest and exceeded those of their pentavalent counterparts by several orders of magnitude. Non-methylating cells (urothelial cells and fibroblasts) seem to accumulate higher amounts of arsenic within the cell than the methylating hepatocytes. Cellular uptake and extrusion seem to be faster in hepatocytes than in urothelial cells. The correlation of uptake with toxicity indicates a significant role of membrane permeability towards toxicity. Furthermore, cytotoxic effects are more distinct in hepatocytes. Differential centrifugation studies revealed that elevated concentrations of arsenic are present in the ribosomal fraction of urothelial cells and in nucleic and mitochondrial fractions of hepatic cells. Further studies are needed to define the implications of the observed enrichment of arsenic in specific cellular organelles for its carcinogenic activity. This review summarizes our recent research on cellular uptake, distribution and toxicity of arsenic compounds in methylating and non-methylating cells.

Role of intestinal microbiota in transformation of bismuth and other metals and metalloids into volatile methyl and hydride derivatives in humans and mice.

ABSTRACT The present study shows that feces samples of 14 human volunteers and isolated gut segments of mice (small intestine, cecum, and large intestine) are able to transform metals and metalloids into volatile derivatives ex situ during anaerobic incubation at 37°C and neutral pH. Human feces and the gut of mice exhibit highly productive mechanisms for the formation of the toxic volatile derivative trimethylbismuth [(CH3)3Bi] at rather low concentrations of bismuth (0.2 to 1 μmol kg−1 [dry weight]). An increase of bismuth up to 2 to 14 mmol kg−1 (dry weight) upon a single (human volunteers) or continuous (mouse study) administration of colloidal bismuth subcitrate resulted in an average increase of the derivatization rate from approximately 4 pmol h−1 kg−1 (dry weight) to 2,100 pmol h−1 kg−1 (dry weight) in human feces samples and from approximately 5 pmol h−1 kg−1 (dry weight) to 120 pmol h−1 kg−1 (dry weight) in mouse gut samples, respectively. The upshift of the bismuth content also led to an increase of derivatives of other elements (such as arsenic, antimony, and lead in human feces or tellurium and lead in the murine large intestine). The assumption that the gut microbiota plays a dominant role for these transformation processes, as indicated by the production of volatile derivatives of various elements in feces samples, is supported by the observation that the gut segments of germfree mice are unable to transform administered bismuth to (CH3)3Bi.

Subcellular Distribution of Inorganic and Methylated Arsenic Compounds in Human Urothelial Cells and Human Hepatocytes

Epidemiological studies have indicated that exposure of humans to inorganic arsenic in drinking water is associated with the occurrence of bladder cancer. The mechanisms by which arsenic induces this malignancy are still uncertain; however, arsenic metabolites are suspected to play a pivotal role. The aim of the present study was the investigation of uptake capabilities of human urothelial cells (UROtsa) compared with primary human hepatocytes (phH) as well as the intracellular distribution of the arsenic species. Additionally, we were interested in the cyto- and genotoxic potential (comet assay, radical generation) of the different arsenic compounds in these two cell types. Our results show that UROtsa cells accumulate higher amounts of the arsenic species than the phH. Differential centrifugation revealed that the arsenic compounds are preferentially distributed into nuclei and ribosomes. After 24-h exposure, arsenic is mainly found in the ribosomes of UROtsa cells and in the nuclei and mitochondria of phH. In contrast to the pentavalent arsenic species, the trivalent species induced a 4- to 5-fold increase of DNA damage in hepatocytes. Radical generation, measured by thiobarbituric acid reactive substances, was more pronounced in hepatocytes than in urothelial cells. In summary, the uptake of arsenic compounds appears to be highly dependent upon cell type and arsenic species. The nonmethylating urothelial cells accumulate higher amounts of arsenic species than the methylating hepatocytes. However, cyto- and genotoxic effects are more distinct in hepatocytes. Further studies are needed to define the implications of the observed accumulation in cellular organelles for the carcinogenic activity of arsenic.

Methylated Bismuth, but Not Bismuth Citrate or Bismuth Glutathione, Induces Cyto- and Genotoxic Effects in Human Cells in Vitro

Bismuth compounds are widely used in industrial processes and products. In medicine, bismuth salts have been applied in combination with antibiotics for the treatment of Helicobacter pylori infections, for the prevention of diarrhea, and in radioimmunotherapy. In the environment, bismuth ions can be biotransformed to the volatile bismuth compound trimethylbismuth (Me3Bi) by methanobacteria. Preliminary in-house studies have indicated that bismuth ions are methylated in the human colon by intestinal microflora following ingestion of bismuth-containing salts. Information concerning cyto- and genotoxicity of these biomethylated products is limited. In the present study, we investigated the cellular uptake of an organic bismuth compound [monomethylbismuth(III), MeBi(III)] and two other bismuth compounds [bismuth citrate (Bi-Cit) and bismuth glutathione (Bi-GS)] in human hepatocytes, lymphocytes, and erythrocytes using ICP-MS. We also analyzed the cyto- and genotoxic effects of these compounds to investigate their toxic potential. Our results show that the methylbismuth compound was better taken up by the cells than Bi-Cit and Bi-GS. All intracellularly detected bismuth compounds were located in the cytosol of the cells. MeBi(III) was best taken up by erythrocytes (36%), followed by lymphocytes (17%) and hepatocytes (0.04%). Erythrocytes and hepatocytes were more susceptible to MeBi(III) exposure than lymphocytes. Cytotoxic effects of MeBi(III) were detectable in erythrocytes at concentrations >4 microM, in hepatocytes at >130 microM, and in lymphocytes at >430 microM after 24 h of exposure. Cytotoxic effects for Bi-Cit and Bi-GS were much lower or not detectable in the used cell lines up to a tested concentration of 500 microM. Exposure of lymphocytes to MeBi(III) (250 microM for 1 h and 25 microM/50 microM for 24 h) resulted in significantly increased frequencies of chromosomal aberrations (CA) and sister chromatid exchanges (SCE), whereas Bi-Cit and Bi-GS induced neither CA nor SCE. Our study also showed an intracellular production of free radicals caused by MeBi(III) in hepatocytes but not in lymphocytes. These data suggest that biomethylation of bismuth ions by the intestinal microflora of the human colon leads to an increase in the toxicity of the primary bismuth salt.

Environmental exposure, chlorinated drinking water, and bladder cancer

Environmental and/or occupational factors have been proposed to play a critical role in urological malignancies and, in particular, in bladder cancer. Epidemiological studies have demonstrated with sufficient evidence that factors such as smoking and exposure to aromatic amines, paints and solvents, leather dust, inks, some metals, polycyclic aromatic hydrocarbons, combustion products, or diesel exhaust fumes are associated with the development of bladder cancer. Candidates with an uncertain potential for inducing this type of cancer include dietary factors, specifically fats and cholesterol, and the exposure to contaminants in drinking water. This chapter will describe and discuss the respective literature on environmental and occupational factors linked to carcinogenesis in bladder cancer. For several reasons, the potential effects of tea and coffee consumption will also be considered. A solid epidemiological evaluation of environmental and occupational factors linked to carcinogenesis has to meet many challenges: the number of confounding factors is often large, exposure needs to be determined retrospectively, and elevation of the attributable risk is low in most cases. In view of the long-term exposure of the vast majority of the population to, for instance, drinking- water contaminants, however, the impact of even small elevations of risk warrants evaluation. This complex task needs comprehensive approaches on a large scale including modern analytical, molecular biological and epidemiological methods.

Determination of Trimethylbismuth in the Human Body after Ingestion of Colloidal Bismuth Subcitrate

Biological methylation and hydride formation of metals and metalloids are ubiquitous environmental processes that can lead to the formation of chemical species with significantly increased mobility and toxicity. Whereas much is known about the interaction of metal(loid)s with microorganisms in environmental settings, little information has been gathered on respective processes inside the human body as yet. Here, we studied the biotransformation and excretion of bismuth after ingestion of colloidal bismuth subcitrate (215 mg of bismuth) to 20 male human volunteers. Bismuth absorption in the stomach and upper intestine was very low, as evidenced by the small quantity of bismuth eliminated via the renal route. Total bismuth concentrations in blood increased rapidly in the first hour after ingestion. Most of the ingested bismuth was excreted via feces during the study period. Trace levels of the metabolite trimethylbismuth [(CH3)3Bi] were detected via low temperaturegas chromatography/inductively coupled plasma-mass spectrometry in blood samples and in exhaled air samples. Concentrations were in the range of up to 2.50 pg/ml (blood) and 0.8 to 458 ng/m3 (exhaled air), with high interindividual variation being observed. Elimination routes of bismuth were exhaled air (up to 0.03‰), urine (0.03–1.2%), and feces. The site of (CH3)3Bi production could not be identified in the present study, but the intestinal microflora seems to be involved in this biotransformation if accompanying ex vivo studies are taken into consideration.

14:Methylated Metal(loid) Species in Humans

While the metal(loid)s arsenic, bismuth, and selenium (probably also tellurium) have been shown to be enzymatically methylated in the human body, this has not yet been demonstrated for antimony, cadmium, germanium, indium, lead, mercury, thallium, and tin, although the latter elements can be biomethylated in the environment. Methylated metal(loid)s exhibit increased mobility, thus leading to a more efficient metal(loid) transport within the body and, in particular, opening chances for passing membrane barriers (blood-brain barrier, placental barrier). As a consequence human health may be affected. In this review, relevant data from the literature are compiled, and are discussed with respect to the evaluation of assumed and proven health effects caused by alkylated metal(loid) species.

Organometal(loid) compounds associated with human metabolism

Biomethylation of metals and metalloids is a well-known process ubiquitously occurring in the environment, which leads to the formation of chemical species with significantly higher mobility and altered toxicology. There are only a few historical reports, e.g. about “bismuth breath” or “Gosio gas” dealing with the association of humans with methylated metal(loid)s. Although the toxicity of the latter [later identified as trimethyl arsine (Challenger 1945)] has not been conclusively demonstrated, this gas produced by fungi in wet wallpaper was considered to be the reason for the illness of people living there (Gosio 1897). Amongst other observations, dimethyltellurium in “bismuth breath” of mine workers, dimethylselenium in the upper ng/m3 range in human breath, as well as the detection of at least twenty-two different organometal(loid) species in human urine are indications for the methylation of metal(loid)s occurring in humans (Cai et al. 1995; Feldmann et al. 1996; Kresimon et al. 2001).