Environ. Sci. Technol. 2003, 37, 845-851
Metabolism of Arsenic by Sheep Chronically Exposed to Arsenosugars as a Normal Part of Their Diet. 1. Quantitative Intake, Uptake, and Excretion H. R. HANSEN,† A. RAAB,† K. A. FRANCESCONI,‡ AND J . F E L D M A N N * ,† Environmental Analytical Chemistry, Department of Chemistry, University of Aberdeen, Meston Walk, Old Aberdeen AB24 3UE, U.K., and Institute of Chemistry-Analytical Chemistry, Karl-Franzens University, 8010 Graz, Austria
Information on the effects of long-term organoarsenical consumption by mammals is limited despite the fact that foodstuffs, especially seafood, often contain organoarsenicals at very high concentrations. Here we evaluate the intake, uptake, and excretion (urine and feces) of arsenic by sheep that live on North Ronaldsay in the Orkney Islands and naturally consume large amounts of arsenosugars through their major food sourcesseaweed. The sheep eat a broad variety of seaweed species, and arsenic concentrations were determined in all the species observed eaten by the sheep (5.7-74.0 mg kg-1 dry mass). Because of preference and availability, they feed mostly on the seaweed species found to contain the highest arsenic concentrations: Laminaria digitata and Laminaria hyperborea (74 ( 4 mg kg-1 dry mass). To quantify the arsenic intake by the sheep, a feeding experiment reflecting natural conditions as close as possible was set up. In the feeding trial, the average daily intake of arsenic by 12 ewes was 35 ( 6 mg (97% of water-extractable arsenic was present as arsenosugars) gained from feeding on the two brown algae. To test the possible influence of microflora on the metabolism of arsenosugars, six of the sheep were adapted to feeding on grass for 5 months before the start of the trial (control sheep), and the remaining six sheep were kept on their normal seaweed diet (wild sheep). No significant difference in seaweed/arsenic intake and arsenic excretion was found between the two groups of sheep. The arsenic excreted in the feces represents 13 ( 10% (n ) 12) of the total consumed, and on the assumption of that, the average urinary excretion is estimated to 86%.The main arsenic metabolite excreted in urine was dimethylarsinic acid (DMA(V)) (60 ( 22%) and minor amounts of dimethylarsinoylethanol (DMAE), methylarsonic acid (MA(V)), tetramethylarsonium ion (TMA+), and arsenate (As(V)) together with seven unknown arsenic compounds were also excreted. The urinary arsenic excretion pattern showed a lag period (>4 h) * Corresponding author telephone: (0)1224-272911; fax: (0)1224272921; e-mail:
[email protected]. † University of Aberdeen. ‡ Karl-Franzens University. 10.1021/es026074n CCC: $25.00 Published on Web 01/30/2003
2003 American Chemical Society
before significant quantities appeared in the urine, an excretion rate that peaked between 4 and 28 h after seaweed intake and a relatively slow half-life (17 h) after end of intake.
Introduction Millions of people in the world are exposed to significant quantities of inorganic arsenic, primarily through their drinking water. This exposure contributes to cancers of skin, bladder, and lung in addition to having noncancer health effects (1). The toxicity of arsenic is highly dependent on its chemical form, and most research has been on exposure to inorganic arsenic as it was believed to be more toxic than organic arsenic species. The metabolic pathway of inorganic arsenic is well-documented (2, 3). Humans are mainly exposed to organoarsenicals through seafood; marine animals such as fish and molluscs are generally rich in arsenobetaine [(CH3)3As+CH2COO-], and marine algae accumulate inorganic arsenic from seawater and incorporate it into a range of carbohydrate compounds that are collectively referred to as arsenosugars (more than 100 mg of arsenic kg-1 dry mass; 4). Arsenobetaine is not metabolized in the mammalian body and is excreted unchanged in urine within a few hours after intake (5, 6). The fact that arsenobetaine is rapidly excreted in the urine without metabolic change may have led to the perception that all organoarsenicals of seafood origin are excreted unchanged. Arsenosugars however show an excretion pattern quite different from that displayed by arsenobetaine. Arsenosugars are, like inorganic arsenic, largely metabolized in mammals, and the metabolites are excreted relatively slowly. Individual variation in urinary arsenic excretion after intake of inorganic arsenic by humans is known, and 46-66% of a dose has been reported to be excreted within a few days (7-9). The corresponding value for a chemically synthesized arsenosugar is 80% during 4 days (10). The major metabolite excreted in both cases is DMA(V) (3, 10-12). The exposure of humans to arsenosugars is by tradition relatively high in the Asian world because of the frequent use of seaweed in cooking. In the Western Hemisphere, the exposure is increasing because of the introduction of Asian cooking habits and the increasing use of seaweed as a health product (13). On North Ronaldsay (the smallest of the Orkney islands, north of Scotland), the population of native sheep are restricted to a life on the seashore and feed almost exclusively on seaweed (14). Because of their arsenic-rich seaweed diet, these sheep have the highest arsenosugar intake known by any mammal; hence, they offer an excellent opportunity to study the arsenosugar metabolism in a mammalian organism. Only a few long-term feeding studies have reported the disposition of arsenic in tissues of mammals, and these studies deal mostly with inorganic arsenic (15-19). Limited information is available on arsenic accumulation in tissues of mammals after long-term consumption of arsenosugars. In a previous study on the North Ronaldsay sheep, elevated arsenic concentrations were measured in liver (0.29 ( 0.10 mg of As kg-1 dry mass), kidney (0.57 ( 0.19 mg of As kg-1 dry mass), and muscle (0.68 ( 0.22 mg of As kg-1 dry mass) (12). The arsenic concentrations in the North Ronaldsay sheep were elevated more than 100 times as compared with tissue concentrations of sheep with a normal diet (20); after arsenosugar intake, arsenic does accumulate to a certain degree. VOL. 37, NO. 5, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Structures of the four arsenosugars most often identified in the environment and investigated work. The arsenosugars themselves are currently considered to be nontoxic (21, 22). However, because the disposition of arsenosugars shows similarities to the disposition of inorganic arsenic (in terms of the main biotransformation product DMA(V) and accumulation) and because toxic intermediate metabolites may be present in the biotransformation processes, it is important to study the metabolism of arsenosugars. This paper covers quantitative aspects of arsenosugar metabolism in North Ronaldsay sheep in addition to briefly discussing the major arsenic metabolites identified in urine.
Materials and Methods Experimental Setup. A feeding experiment was carried out with two groups of 6 healthy North Ronaldsay ewes (1-3 years old, average mass 23 ( 5 kg) on North Ronaldsay. One group had been living on the beach and feeding on seaweed for at least 1 year (wild sheep), and the other group had been on grass on the center of the island for 5 months prior to the feeding experiment (control sheep). To simulate natural conditions, the feeding trial was made in a pen on the beach, within which the sheep were corralled in separate 6-m2 boxes. The experiment ran for 11 days with each group; first 11 days with the wild sheep followed immediately by 11 days with the control sheep. On day 1, the sheep were not fed. Each sheep was then fed one meal of a mixture of Laminaria hyperborea and Laminaria digitata (3-5 kg) once a day for days 2-6. The sheep ate most of the seaweed within a few hours. On day 7, the sheep were taken onto grass and kept there for 5 days. All feces were collected from each sheep on days 1-6, and the total wet mass of the feces was recorded once a day (spring scale ( 0.01 g). Fresh seaweed was collected each day and was cleaned of larger epiphytes and epifauna, and its wet mass was recorded (( 0.1 kg). In addition to the seaweed collected for feeding the sheep, a separate batch was collected to correct the seaweed wet mass for its daily evaporation rate (desiccation). Sample Collection and Preparation. All the seaweed species observed eaten by the sheep, grass, and water samples were collected at North Ronaldsay. All samples were frozen (-20 °C) immediately after collection until analysis. Five fronds or branches from five individual specimens each of L. hyperborea, L. digitata, Alaria esculenta, Ascophyllum nodosum, Chorda filum, Fucus spiralis, Himanthalia elongata, L. saccharina, Pelvetia canaliculata, Saccorhiza polyschides, Codium fragile, Enteromorpha intestinalis, Ulva 846
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FIGURE 2. Anion-exchange chromatogram of arsenic standards; arsenite (As(III)), arsenosugar 1, dimethylarsinic acid (DMA(V)), methylarsonic acid (MA(V)), arsenosugar 2, arsenate (As(V)), and arsenosugar 3, mobile phase pH 5.3 (A). Cation-exchange chromatograms of arsenic standards; As(V), As(III), DMA(V), arsenobetaine (AsB), arsenosugar 1, arsenocholine (AC), and tetramethylarsonium ion (TMA+), mobile phase pH 3.0 (B), and of DMAE and TMAO, mobile phase pH 2.5 (C). lactuca, and Palmaria palmata were collected from the seaweed biomass recently washed ashore. The samples were rinsed in seawater and frozen. Grass was collected from the paddock used in the feeding experiment, and water was sampled from three different drinking sources used by the sheep. Urine samples were collected twice a day in “nappies” specially designed to fit the sheep without hampering them. The nappies were placed on the sheep for 1-2 h before each urine collection. Urine was squeezed out of the nappy, transferred to polyethylene tubes, and stored in a refrigerator at 5 °C. The aim was to collect urine samples 4 and 20 h after the sheep were fed a seaweed meal. However, because of the method of sample collection, this was not always possibly, and the exact collection time of each individual varied within a few hours. Feces were collected fresh (
