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. served in arsenocholine dosing experiments. At 2 h, 61 and 16% of the administered arsenic were recovered in viscera Speciation of Arsenic and muscle, respectively. As a result of subsequent trans The minced tissue was extracted three times with 3 portation of arsenic from viscera to muscle, the amount of volumes of methanol and the extract evaporated to dry arsenic retained in muscle exceeded that in viscera at 72 h. ness. The residue was suspended in distilled water (20-40 ml) and shaken three times with an equal volume of Chemical Form of the Arsenic Retained by Carp diethyl ether to remove lipids. The aqueous phase (water The following viscera and muscle were used for specia soluble fraction) was concentrated to about 10 ml and ap tion of the retained arsenic: viscera at 2 and 72 h for all plied to a Dowex 50X2 column (100-200 mesh, H+ form, arsenic compounds, muscle at 2 h for arsenate , arseno 2 x 20 cm), which was eluted successively with 300 ml each betaine, and arsenocholine, and muscle at 72 h for ar- Metabolism of Arsenic Compounds in Carp 263

Fig. 2. HPLC-ICP/AES analysis of the unadsorbed fraction in ar senate dosing to carp. HPLC conditions: column, Nucleosil 10SB (0.46 x 25 cm); sol vent, 0.01 M phosphate buffer (pH 7.0); flow rate, 1 ml/min. Stan dard arsenic compounds: As (‡V), arsenite; MMA, monomethylar sonate; As (V), arsenate.

the majority (80-99%) of the arsenic in the water-soluble fraction appeared in the unadsorbed fraction for arsenate and in the NH4OH fraction for the other four compounds. The only exception was observed with the muscle at 2 h in arsenocholine dosing experiments; the NH4OH and HCl Fig. 1. Retention of arsenic in carp after intraperitoneal administration fractions accounted for 43 and 51% of the arsenic in the of arsenic compounds. water-soluble fraction, respectively. Thus, the unadsorbed •¡, viscera; •£, muscle; •œ, viscera+muscle. fraction for arsenate, the NH4OH fraction for dimethyl arsinate, arsenobetaine or trimethylarsine oxide, and both NH4OH and HCl fractions for arsenocholine were ana senobetaine and arsenocholine. For these tissues, 75-95% lyzed by HPLC-ICP/AES. of the retained arsenic was extracted with methanol and In arsenate dosing experiments, the unadsorbed fraction recovered in the water-soluble fraction. of the viscera at 2 h gave two arsenic peaks corresponding The behaviors on Dowex 50 of arsenic compounds in to arsenite and arsenate, while those of the viscera at 72 h the water-soluble fraction are summarized as follows. and the muscle at 2 h gave only one peak corresponding to Regardless of the tissue and the time elapsed after dosing, arsenite, suggesting the reduction of arsenate to arsenite in 264 Shiomi et al.

Fig. 3. HPLC-ICP/AES analysis of the NH4OH fraction in dimethylar Fig. 4. HPLC-ICP/AES analysis of the NH4OH fraction in ar sinate dosing to carp. senobetaine dosing to carp. HPLC conditions as in Fig. 2. Standard arsenic compounds: AB, HPLC conditions: column, Chemcosorb 7SCX (0.46 X 25 cm); sol arsenobetaine; TMAO, trimethylarsine oxide; DMA, dimethylar vent, 0.05 Mpyridine-formate buffer (pH 3.I); flow rate, 1 ml/min. sinate. Standard arsenic compounds as in Fig. 3.

carp (Fig. 2). In the case of dimethylarsinate dosing, only With respect to the retention of arsenic in carp, the five the dosed compound was observed in the NH4OH fraction arsenic compounds used are classified into two types. Low of the viscera at 2 and 72 h (Fig. 3). Similarly, administra retention was found for arsenate, dimethylarsinate, and tion of arsenobetaine resulted in the detection of only trimethylarsine oxide and high retention for arsenobetaine arsenobetaine in the NH4OH fraction of either viscera or and arsenocholine. The results obtained with arsenate, muscle (Fig. 4). In the case of trimethylarsine oxide dos arsenobetaine, and arsenocholine are essentially the same ing, the NH4OH fraction of viscera contained two com as in the case of their oral administration to fish.5,6)Also, pounds, trimethylarsine oxide and another compound the result with dimethylarsinate may be comparable to the (probably dimethylarsinate); the ratio of the latter com previous finding that three dimethylated arsenic com pound to the former increased with time (Fig. 5). Biotrans pounds (2-dimethylarsinylethanol, 2-dimethylarsinyl formation was also found in arsenocholine dosing experi acetate, and 2-dimethylarsinothioylethanol) are scar ments, in which arsenobetaine was found in the NH4OH cely retained by fish when orally administered.) Thus, it is fraction of both viscera and muscle even at 2 h (Fig. 6) very likely that the retention or excretion of a given arsenic while arsenocholine was found in the HCl fraction (Fig. 7). compound is independent of its administration route, although no information is available as to the retention of Discussion arsenic upon oral administration of trimethylarsine oxide. Arsenate and arsenocholine are partly or largely convert In this study, carp were chosen as experimental fish be ed to arsenite and arsenobetaine, respectively, within the cause of their low arsenic concentrations (0.4ƒÊgAs/g in carp. Trimethylarsine oxide is also converted to another muscle and 0.3ƒÊgAs/g in viscera) similar to other compound (probably dimethylarsinate). In contrast, no freshwater fish as well as because of their ready availabil biotransformation of dimethylarsinate and arseno ity. As a result, the retention and biotransformation by the betaine occurs. The reduction of arsenate to arsenite carp of the arsenic compounds administered at high levels by rainbow trout has previously been suggested by could be examined without being affected by the arsenic Oladimeji et al.4) Edmonds and Francesconi5l overlooked originally contained in the fish. this reduction in estuary catfish and school whiting but in Metabolism of Arsenic Compounds in Carp 265

Fig. 5. HPLC-ICP/AES analysis of the NH4OH fraction in Fig.-6. HPLC-ICP/AES analysis of the NH4OH fraction in arsenocho trimethylarsine oxide dosing to carp. line dosing to carp. HPLC conditions as in Fig. 4 and standard arsenic compounds as HPLC conditions as in Fig. 4 and standard arsenic compounds as in Fig. 3. in Fig. 3.

stead found that oral administration of arsenate resulted is useful to summarize the current information about the in the accumulation of trimethylarsine oxide in the fish. metabolism of arsenic compounds in fish as follows. Ar On the other hand, the possible demethylation of senate, dimethylated compounds, and trimethylarsine trimethylarsine oxide to dimethylarsinate is of interest, oxide are scarcely retained by fish, while arsenobetaine since it is generally considered that methylation progresses and arsenocholine are largely accumulated. Biotransforma in animals to detoxify arsenic compounds. Further tion by fish is observed for three compounds; arsenate is rigorous identification by spectral analyses of the com converted to arsenite and/or trimethylarsine oxide, pound formed following intraperitoneal administration of trimethylarsine oxide to an unknown compound (prob trimethylarsine oxide is needed to clarify definitively ably dimethylarsinate), and arsenocholine to arseno whether demethylation of trimethylarsine oxide occurs in betaine. Current information suggests that the reten fish or not. The oxidation of arsenocholine to arseno tion of arsenic compounds by fish is distinct from that by betaine has already been established by Francesconi et mammals, although no significant difference in their a1.,6) who studied the fate of arsenocholine orally ad biotransformation is recognized between fish and mam ministered to yelloweye mullet. However, it should be mals. Arsenate is readily excreted by fish, while its elimina emphasized that this study was designed to clarify the tion from mammals is rather slow compared with organo metabolism of arsenic compounds by fish themselves, arsenicals.9a On the contrary, the arsenic administered namely by enzymes in the fish tissues, not by the bacterial as either arsenobetaine or arsenocholine accumulates in action in the fish gut tract previously assumed for the con fish but is rapidly excreted by mammals.10-13) version of orally administered arsenate3,5) or arseno Further metabolic studies are needed to clarify the de choline.6) It is especially important to examine whether tailed mechanism of accumulation of some arsenic com bacteria in the fish gut tract easily metabolize arsenocholine pounds such as arsenobetaine in fish. Especially valuable to arsenobetaine. information may be provided by metabolic studies using At present the metabolism of arsenic compounds in fish arsenosugars, the major arsenic compounds in algae. is not fully understood. Only limited species of fish have been used in studies. In addition, arsenic compounds were administered intraperitoneally to fish in our study, while other studies adopted oral administration. Nevertheless, it 266 Shiomi et al.

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