Kazuo Shiomi, Yasuo Sugiyama, Kuniyoshi Shimakura, and Yuji Nagashima
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Fisheries Science 62(2), 261-266 (1996) Retention and Biotransformation of Arsenic Compounds Administered Intraperitoneally to Carp Kazuo Shiomi, Yasuo Sugiyama, Kuniyoshi Shimakura, and Yuji Nagashima Department of Food Science and Technology, Tokyo University of Fisheries, Konan, Minato, Tokyo 108, Japan (Received June 13, 1995) The arsenic metabolism in fish was examined using carp Cyprinus carpio and five arsenic com pounds (arsenate, dimethylarsinate, arsenobetaine, trimethylarsine oxide, and arsenocholine). In order to avoid the bacterial action in the gut tract suggested previously, the arsenic compounds were ad ministered to the fish not orally but intraperitoneally. Low retention of arsenic by the fish was observed after administration of arsenate, dimethylarsinate, or trimethylarsine oxide, while the arsenic ad ministered as either arsenobetaine or arsenocholine was highly retained. After extraction and partial purification by Dowex 50 column chromatography, arsenic compounds accumulated in viscera and mus cle were analyzed by HPLC-ICP/AES. As a result, arsenate and arsenocholine were found to be con verted to arsenite and arsenobetaine, respectively, within the fish. Conversion of trimethylarsine oxide to another compound (probably dimethylarsinate) was also observed. In contrast, no biotransforma tion of dimethylarsinate and arsenobetaine occurred. Key words: arsenic compounds, carp, retention, biotransformation, metabolism Marine animals accumulate significant amounts of arse periments using yelloweye mullet Aldrichetta forsteri, no nic (at the ppm level) in the organic form. The major or sole retention of dimethylated arsenic compounds was ob organoarsenic compound in a variety of marine animals served, while the arsenic administered as either arseno has been shown to be arsenobetaine; in very limited spe betaine or arsenocholine was highly retained by the cies, other organoarsenic compounds such as arseno fish.6) Although arsenobetaine was accumulated in the fish choline and trimethylarsine oxide have also been detected, without conversion, arsenocholine was readily converted usually as minor constituents.1) These organoarsenic com to arsenobetaine by the fish. pounds are considered to pass to marine animals mainly As described above, arsenate and arsenocholine, when through the food chain. Within the food chain, however, orally administered, are converted to other forms by fish. biotransformation of arsenic by marine animals must oc It is, however, suggested that the observed conversion is cur, since marine algae which are at the primary stage of possibly dependent on the bacterial action of the fish gut the food chain are devoid of the organoarsenic com tract.3,5,6) Whether arsenic compounds can be biotrans pounds found in marine animals and instead contain more formed by fish themselves remains to be resolved. It complex compounds, arsenosugars (arsenic-containing seems to be unlikely that a compound administered intra ribosides), as the major arsenicals.1) These circumstances peritoneally appears in the gut tract and then its bacteri are probably true for the freshwater environment, in con al metabolites are again absorbed from the gut tract. In sideration of our recent finding that arsenobetaine is also this study, therefore, five arsenic compounds (arsenate, the major arsenic compound in freshwater fish.2) For a bet dimethylarsinate, arsenobetaine, trimethylarsine oxide, ter understanding of the accumulation mechanism of some and arsenocholine) were administered intraperitoneally to arsenic compounds such as arsenobetaine in aquatic carp Cyprinus carpio and their retention and biotrans animals, it is essential to clarify the arsenic metabolism in formation by the fish were examined. Among the five ar them. senic compounds, dimethylarsinate and trimethylarsine The metabolism of several arsenic compounds by fish oxide have not been dealt with in any previous metabolic have been studied, including both marine and freshwater studies using oral administration to fish. fish. Penrose3) showed that sodium arsenate administered orally to brown trout Salmo trutta was rapidly converted Materials and Methods to organic forms. Similar results were also obtained with rainbow trout Salmo gairdneri by Oladimeji et al.4) The Carp metabolism of arsenic compounds by fish was later exam Juvenile carp (6 months old; total length 15.7-18.8 cm, ined in more detail by Edmonds and co-workers.5,6) Oral body weight 45-83 g), which had been hatched at the administration of sodium arsenate to estuary catfish Yoshida Research and Training Station, Tokyo University Cnidoglamis macrocephalus or school whiting Sillago of Fisheries, located in Shizuoka Prefecture, were trans bassensis resulted in the formation of trimethylarsine oxide, ported alive to our laboratory. They were maintained in although the retained arsenic was at a trace level.5) In ex- oxygenated tap water without diets for at least 72 h before 262 Shiomi et al. the start of experiments. of distilled water (unadsorbed fraction), 1 M NH4OH (NH4OH fraction), distilled water, and 1 M HCl (HCl frac Reagents tion). Arsenic-rich fractions were concentrated to dryness. Nitric acid (containing 61% HNO3), perchloric acid The dried material was dissolved in 1 ml of distilled water (containing 60% HCIO4), and sulfuric acid (containing and filtered through a 0.45ƒÊm membrane. The filtrate was 97% H2SO4) used for wet-digestion were of super special analyzed by the HPLC-ICP/AES system, essentially ac grade. Sodium arsenate, sodium arsenite, and dimethylar cording to the method of Shiomi et al.7) Briefly, a sinate were purchased from Wako Pure Chemical Co. Nucleosil IOSB column (0.46 x 25 cm; Macherey-Nagel, (Tokyo, Japan) and sodium monomethylarsonate from Germany) with 0.01 M phosphate buffer (pH 7.0) or a Ventron Corp. (Beverly, MA, U.S.A.). Synthetic ar Chemcosorb 7SCX column (0.46 x 25 cm; Chemco, senobetaine, trimethylarsine oxide, arsenocholine, and Takatsuki) with 0.05 M pyridine-formate buffer (pH 3.1) tetramethylarsonium iodide were kindly donated by Dr. T. was used for HPLC. The eluate from the column was con Kaise, Tokyo College of Pharmacy. The other reagents tinuously introduced to the nebulizer of the ICP/AES and were of analytical grade. its arsenic concentration was read at 10 s intervals. In the HPLC-ICP/AES analysis of each fraction, the most suita Determination of Arsenic ble standard arsenic compounds were selected, based on Solid samples were accurately weighed into a 100 ml the fact that arsenate, arsenite, and monomethylarsonate beaker. To the beaker were added 25 ml of nitric acid, 5 ml are eluted in the unadsorbed fraction from Dowex 50, of perchloric acid, and 0.5 ml of sulfuric acid. The beakeeer dimethylarsinate, arsenobetaine, and trimethylarsine was covered with a watch glass and kept at room tempera oxide in the NH4OH fraction and arsenocholine and ture overnight in order to avoid the vigorous reaction in tetramethylarsonium iodide in the HCl fraction.7,8) duced by immediate heating. Then the beaker was heated at about 200•Ž in a draft chamber. After wet-digestion Results and evaporation of the acids, the beaker was washed with distilled water and the washing made up to 10 ml in a volu Retention of Arsenic by Carp metric flask. This solution was passed through a filter The retention profiles of arsenic in carp following intra paper and the filtrate measured for arsenic with an induc peritoneal administration of five arsenic compounds are tively coupled argon plasma emission spectrometer (ICP / illustrated in Fig. 1. Arsenic concentrations of muscle and AES; Jarrell-Ash AtomComp Series 800) under the follow viscera pooled from three control fish receiving no arsenic ing conditions: argon flow rate, nebulizer 0.45 //min, aux were 0.4 and 0.3 ƒÊgAs/g, respectively. The retained arse iliary 0.3 1/min, plasma 71/min; wavelength, 193.7 nm; ra nic in carp was calculated by subtracting the above values dio-frequency power, 1.25 kW; observation height, 16 for the control fish from those estimated for the experimen mm; and integration time, 20s. The ICP/AES was tally dosed fish. In addition, the sum of the arsenic calibrated using distilled water and sodium arsenate solu retained in muscle and viscera was assumed to be the total tion (10ƒÊgAs/ml) made in distilled water. For aqueous arsenic retained in the fish. samples, their arsenic concentrations were directly esti Low retention of arsenic was observed for three arsenic mated on the ICP/AES without wet-digestion. compounds (arsenate, dimethylarsinate, and trimethyl arsine oxide). When these three compounds were ad Arsenic Dosing Experiments ministered, about 75% and more than 90% of the ad Arsenate, dimethylarsinate, arsenobetaine, trimethylar ministered arsenic were eliminated from the fish at 2 and sine oxide or arsenocholine was dissolved in distilled water 72 h, respectively. In contrast, administration of either at 1 mgAs/ml. Each solution was injected intraperitoneal arsenobetaine or arsenocholine resulted in high retention of ly at 0.01 ml/g, equivalent to 0.01 mgAs/g, into a group arsenic. For both compounds, more than 65% of the ad of 9 fish, which was kept in a 20l aquarium with ministered arsenic was retained in carp even 72 h after ad oxygenated tap water. Three fish were withdrawn from the ministration. In the case of arsenobetaine, 55 and 10% of group at 2, 24 or 72 h after injection and placed into iced the administered arsenic appeared in muscle and viscera at water to anesthetize them. Muscle and viscera were individ 2 h, respectively, suggesting a rapid transportation of ar ually pooled from the 3 fish and minced with a blender. A senic from viscera to muscle. The amount of arsenic small portion (5 g for muscle and 0.5 g for viscera) of the retained in muscle gradually increased with time, while minced tissue was measured for total arsenic. For the speci that in viscera decreased. On the other hand, a slower ation of arsenic, 20 g of muscle and 12-19 g of viscera transportation of arsenic from viscera to muscle was ob were used.