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1 A~=hivel of Arch. Environ. Contam. Toxicol. 13, (1984) E nvironrnenl=~ Tox o Springer-Verlag New York Inc. Tissue Distribution and Retention of 74As-Dimethylarsinic Acid in Mice and Rats 1 Marie Vahter.2, Erminio Marafante**, and Lennart Dencker? * National Institute of Environmental Medicine, P.O. Box 60208, S Stockholm, Sweden, ** Radiochemistry Division, CEC Joint Research Centre, Ispra, Italy, and tdepartment of Toxicology, Uppsala University, P.O. Box 573, S Uppsala, Sweden Abstract. The metabolism of dimethylarsinic acid (DMA), a common pesticide and the primary metabolite of inorganic arsenic in mammals, has been studied in mice and rats. About 80% of an oral dose (0.4 mg As/kg body weight) was absorbed from the gastrointestinal tract. In the mice, more than 99% of the dose was eliminated within 3 days, as compared to about 50% in the rats, mainly due to accumulation in the blood. The tissue distribution in the mice was characterized by highest initial (0.5-6 hr) concentrations in kidneys, lungs, intestinal mucosa, stomach, and testes. Tissues with longest retention time were lungs, thyroid, intestinal walls and lens. No demethylation of the 74As-DMA to inorganic arsenic was observed, but some of the 74As-DMA in the tissues was apparently in a complexed form. Dimethylarsinic acid (cacodylic acid, DMA) and its sodium salt are widely used as herbicides on noncrop areas and as cotton defoliants (USEPA 1975). It occurs often naturally at low levels in the environment due to methylation of inorganic arsenic by microorganisms and algae (Braman 1975; Andreae 1979; Tagawa 1980). DMA is also the main metabolite of inorganic arsenic in most mammals (Charbonneau et al. 1979, 1980; Tam et al. 1979; Bertolero et al. 1981; Buchet et al. 1981; Vahter 1981). 1 This work was performed within the framework of the contract of collaboration No TS ISP S between the Commission of the European Communities and the National Institute of Environmental Medicine, Stockholm 2 To whom correspondence should be addressed Although DMA is considered less toxic than inorganic arsenic, acute effects including irritation of gastrointestinal mucosa, eyes and skin have been reported following occupational exposure (Peoples et al. 1979). Systemic toxic effects of high doses of DMA have been induced in pregnant mice and rats as well as their fetuses (Harrison et al. 1980; Rogers et al. 1981). Whether the toxic effects of DMA are caused by the compound as such or by inorganic arsenic following demethylation in vivo is not clear. Due to the strong bonds between the arsenic atom and the methyl groups, DMA is very stable toward chemical degradation and is not decomposed by hydrochloric or nitric acid even upon heating (USEPA 1975), but microbial demethylation has been demonstrated (Woolson 1977; Sanders 1979). The therapeutic action of drugs containing sodium cacodylate has been ascribed to the inorganic arsenic produced by demethylation in the tissues (Goodman and Gilman 1955; Martindale 1977). However, DMA administered to man (Buchet et al. 1981), mice and rabbits (Vahter and Marafante 1983) was found to be excreted unchanged in the urine, and Stevens et al. (1977) could not detect any significant demethylation of 14C- and 74As-labelled DMA in rats. Since DMA is the main metabolite of inorganic arsenic, data of its metabolism is of importance also for the evaluation of the tissue distribution and retention of arsenic metabolites following exposure to inorganic arsenic. The tissue distribution of DMA has been reported mainly for rats which in contrast to other species, including man, accumulate arsenic in the red blood cells after exposure to inorganic arsenic (Hunter et al. 1942; Lanz et al. 1950; Marafante et al. 1982; Rowland and Davies 1982) as well as DMA (Stevens et al. 1977; Siewicki 1981).

2 260 Marie Vahter et al Table 1. Elimination of 74As in feces and urine of mice and rats after administration of 74As-DMA (0.4 mg As/kg body weight) orally. Mean values of 4-5 animals _+ S.E. loo! 5O 6 of initialdose Hours after Animal administration Feces Urine rat MICE _ 2.5 (n = 5) RATS _ (n = 4) _+ 0.3 In two cows exposed to 25 mg cacodylic acid with the food daily for 60 days, the main sites of accumulation of arsenic were liver, spleen and pancreas (USEPA 1975). The present paper concerns the fate of 74As-DMA in mice, with special emphasis on the tissue localization, retention, and biotransformation. For comparison the whole-body retention and elimination in rats was also studied. Material and Methods 74As-labelled dimethylarsinic acid (DMA) was obtained from the urine of mice administered 74As-arsenate (Amersham, England). The 74As-metabolites in the 24-hr urine was separated by ion exchange chromatography (AG 50W X4, mesh, Bio- Rad, USA) according to the method of Tam et al. (1978), by which inorganic arsenic, methylarsonic acid and dimethylarsinic acid are sequentially eluted with 0.5M HCI, H20 and 4M NH4OH. After evaporation of the ammonia, the 74As-DMA was further purified on Sephadex LH-20 (Pharmacia, Sweden) in methanol. 74As-DMA analysis by paper electrophoresis (Edwards et al 1975) prior to the administration showed that more than 99% of the 74As was in the form of DMA. Groups of male mice (NMRI, 30 g) and male rats (Sprague Dawley, 200 g) were administered 74As-DMA orally at a dose of 0.4 mg As/kg body weight and placed individually for 2 days in metabolism cages designed for separation of urine (cooled with ice to avoid microbial demethylation of excreted DMA) and feces. The whole-body retention was followed for 4 weeks. The pharmacokinetic parameters of the whole-body clearance were calculated according to the equation C(t~) = C(tt)" exp C-~t) for each linear phase (r 2 > 0.97) of the clearance curve (WHO 1978). Tissue distribution of 74As was studied by whole-body autoradiography and counting of 74As in isolated tissues. Two male C57BL mice (25 g) were intravenously administered 74As-DMA (0.4 mg As/kg body weight, 10 zci/mouse), sacrified by gaseous carbon dioxide after 4 and 24 hr and frozen in a mixture of n-hexane and solid carbon dioxide. Autoradiography was performed according to Ullberg (1954, 1977). Other groups of male mice (NMRI, 30 g) were administered the same dose of 74As-DMA intravenously and sacrificed after different time intervals by cardiac puncture. Blood was collected from the heart and centrifuged at 20,000 rpm for separation of red cells and plasma. Stomach and intestines were cut open and rinsed thoroughly with saline. Other organs were measured for 74As-activity without further treatment. Subcellular fractions of the liver, kidneys and lungs were sep- rio 84 1s O5 ~ mice ~ 30 35days Fig. 1. Whole-body retention of 74As (% of dose) in mice and rats following oral administration of 74As-DMA at a dose of 0.4 mg As/kg body weight. Means + SE of 5 mice and 4 rats arated by differential centrifugation after homogenization in 0.1 M Tris-C1 buffer (ph 8.1) containing 0.25M sucrose. The crude nuclear (10 min at 700 g), mitochondrial (10 min at 9,000 g), lysosomal (25 min at 27,000 g) and microsomal (90 min at 105,000 g) fractions obtained were further purified by two-fold washing followed by centrifugation. Separation of arsenic metabolites in urine, plasma, and soluble cytoplasmic fractions of tissues of mice was performed by ion exchange chromatography on AG 50W X4 as described above after ultrafiltration on Centriflo membranes with a cut-off of 25,000 daltons (Amicon, B.W., Amsterdam, NL). For control, the HC1 and NH3 fractions from the ion exchange chromatography were neutralized and analyzed by paper electrophoresis (Edwards et al. 1975) or, when the 74As-activity was too low for electrophoretic separation, neutralized and passed through the ion exchange columns a second time. A gamma counter (Searle, Nuclear Chicago, Model 1195, with a 3-in diameter NaI/T1/crystal) was used for measurements of 74As in tissues and solutions. For whole-body measurements, a 5 6 in NaI(T1) crystal (Harshaw Chemical, the Netherlands), connected to the gamma counter was used. The amount of 74As-arsenic in the samples was determined by comparison with 74As standard solutions of known specific activity. Results The elimination of 74As in feces and urine of mice and rats up to 48 hr after exposure to 74As-DMA is shown in Table 1. In both species, 13-20% of the oral dose was recovered in the feces within two days, but the elimination was faster in the mice than in the rats. The urinary excretion was considerably lower in the rats than in the mice, the latter showing an almost complete excretion of the absorbed arsenic within two days. Figure 1 shows the whole-body retention of 74As in mice and rats during four weeks after the oral administration. In the mice, about

3 Dimethylarsinic Acid in Mice and Rats 261 Fig. 2. Whole-body autoradiograms of mice after intravenous injection of 74As-DMA (0.4 mg As/kg body weight). A: 4 hr after dosing. B and C: 24 hr after dosing. Note the longterm retention of 74As in the thyroid, the peripheral part of the lens and the intestinal walls (smooth muscles) 85% of the dose was eliminated with a half-time of 2.5 hr, about 14% with a half-time of 10 hr and less than 0.5% with a half-time of 20 days. In the rats, there were only two phases of elimination detected: about 45% of the dose was eliminated with a halftime of about 13 hr, while the remaining 55% was eliminated with a half-time of as much as 50 days. The tissue distribution of 74As in mice following intravenous injection with 74As-DMA as shown by the whole body autoradiography and gamma counting of isolated tissues is illustrated in Figures 2 and 3 and Table 2. The kidneys showed the highest 74As levels 5 to 60 min post-dosing (Table 2, Figure 3). At 4-6 hr, the activity was primarily localized in the lungs, the kidneys, the stomach, and the intestines (also the gastrointestinal content) (Figure 2A and Table 2). The testes also showed fairly high 74As levels (Table 2). The subcellular distribution studies showed that the main part of the cellular 74As was present in the cytosol (Table 3). At 24 hr, there was very little 74As left in the body ( % of the dose) and the activity in the isolated tissues was too low for gamma counting. The whole-body autoradiograms revealed that the 74As was retained particularly in the thyroid, the intestinal wall (smooth muscle layer) and the lens (Figures 2B and C). With ion exchange chromatography, 95 0,9% of the 74As excreted within 48 hr in the mouse urine was eluted with ammonia, similar to the elution of

4 \ 262 Marie Vahter et al. % of dose / g tissue kidneys blood lungs liver 1'o ' 3b ' sb ' m,.u,es Fig. 3. Clearance of 74As from tissues of mice 5-60 min after 74 intravenous injections of As-DMA (0.4 mg As/kg body weight). Means S.E. of 5 mice Table 2. Tissue distribution of 74As in mice after i.v. administration of 74As-DMA (0.4 mg As/kg body weight). Mean S.E. of 3 mice ng As/g tissue Tissue/organ 0.5 hr 6 hr Plasma RBC Brain Epididymis Esophagus Gall bladder Heart Intestine, small Intestine, large Kidneys Liver 222 _ Lungs Skeleton _+ 1.7 Skin Spleen Stomach Thymus Thyroid 151 _ <10 Testis Trachea <10 the 74As-DMA. Further analysis of the neutralized ammonia fractions with paper electrophoresis showed one single spot of 74As at 5 cm in the direction of the anode (+ 5 cm), which corresponded to the mobility of the 74As-DMA standard. The rest of the 74As in the urine (5%) was eluted from the ion exchange resin with 0.5M HCI, suggesting the presence of inorganic arsenic. However, the main HCI peak of 74As appeared in fractions 6-8, compared to fractions 4-5 for the 74As-arsenate standard (Figure 4). When the neutralized HCI fractions were re-chromatographed on the same column all of the 74As was eluted with the ammonia and, when analyzed by paper electrophoresis, 82 _ 9% of the 74As moved similarly to DMA (+ 5 cm). Thus, the 74As in the HC1 fractions was not in the form of inorganic arsenic, but probably in the form of DMAcomplexes, which were hydrolyzed during chromatography. With ion exchange chromatography of the cytosols of liver and kidneys of the mice exposed to 74As- DMA, the percentage 74As eluted with the 0.5M HC1 was higher than for the urine and there were two distinct peaks eluted with the ammonia, especially with the liver cytosols (Figure 4). When the fractions from the ion exchange chromatography containing most 74As were neutralized and re-chromatographed on the AG 50W X4 resin, all of the 74As in the HC1 fractions (peak A) was eluted with the ammonia in fractions 22-24, the 74As in the first ammonia peak (peak B) was partially eluted (50%) with HC1 (fraction 6) and partly with ammonia (fractions 22-24), while all of the 74As of the second ammonia peak (C) was again eluted with the ammonia in fractions 22-24, similar to the elution of the 74As-DMA standard. Thus, the results would indicate the presence of more than one complex of DMA in the tissues. In contrast to the liver and kidney cytosols, essentially all of the 74As in the plasma and the lung cytosol was eluted with the ammonia in the same way as the 74As-DMA standard. Discussion The results indicate that more than 80% of the orally administered 74As-DMA was absorbed from the gastrointestinal tract of both mice and rats. A similar high rate of intestinal absorption of DMA has previously been observed in rats exposed to DMA via the food (4-8 ppm As) for 42 days (Siewicki and Sydlowski 1981). Earlier studies on mice and rats have shown very low fecal excretion following intravenous injection of DMA (Stevens et al. 1977; Vahter and Marafante 1983). The absorbed 74As-DMA in the mice was rapidly excreted in the urine and two days after the administration there was only about 1% of the dose

5 B Dimethylarsinic Acid in Mice and Rats 263 Table 3. Subcellular distribution (% of total homogenate) of 74As in tissues of mice 0.5 and 6 hr after i.v. administration of 74As- DMA (0.4 mg As/kg body weight). 0.5-hr values: means + S.E. of 5 mice; 6-hr values: pooled tissues from 3 mice, single determination cpm ~l~s IIl+V DMA 100 ~,?4As cpm Fraction Liver Kidneys Lungs hr Nuclear _ Mitochondrial < 1 < 1 < 1 Lysosomal < 1 < 1 < 1 Microsomal 0.4 +_ 0.1 <1 <1 Cytosol O-. " Liver 71.6 ~: 7.5 % Lu rigs 100% / ~ / 400- "0 " hr Nuclear Mitochondrial < 1 Lysosomal < 1 Microsomal 1.2 < 1 < 1 Cytosol left in the body. The elimination curve presented in Figure 1 is very similar to that reported for mice exposed to inorganic arsenite or arsenate (Vahter and Norin 1980), which can be explained by the fact that in this species about 80% of the absorbed inorganic arsenic is rapidly methylated to DMA (Vahter 1981). The much lower rate of excretion of the 74As-DMA in the rat, most likely due to the accumulation of DMA in the red blood cells (Stevens et al. 1977), supports the suggestion by Rowland and Davies (1982) that it is primarily the DMA metabolite that is retained in the blood of rats exposed to inorganic arsenic. Since such an accumulation of DMA does not occur in other species, including man, (Charbonneau et al. 1979; Buchet et al. 1981; Vahter and Marafante 1983), it can be concluded that the rat is a poor animal model for studies on the metabolism of DMA as well as of inorganic arsenic. In the mice injected with 74As-DMA, the kidneys had the highest initial concentration of 74As, which agrees with the rapid urinary excretion. Of the other tissues, only the liver, lungs and skin had concentrations similar to that of the plasma. In contrast to the liver and the kidneys where the 7aAs-DMA seemed to be present partly as complexes, essentially all of the 74As in plasma and lungs was in the form of unbound 74As-DMA. The actual forms of the presumed DMA-complexes, which were found in low amounts in the urine as well, have yet to be determined. It has earlier been reported that DMA may react with SH-containing compounds such as 2- mercaptoethanol, cystein, and glutathione (Jacobson et al. 1972). The tissues with the longest retention of 74As were ~ Kidneys % Plasma I00~ -0 0 ~ 0 ~ 30 fractions O.5M HCI 9 H20 9 4MNH 3 9 O.5M HCI 9 H20. 4MNH 3 Fig. 4. Elution profiles of 74As-labelled arsenite, arsenate, and DMA, and 74As in plasma and cytosols of tissues of mice after intravenous injection of 74As-DMA (0.4 mg As/kg body weight); ion exchange chromatography on Bio-Rad AG 50W X4 "0-400 the lungs, the intestinal wails, the thyroid, and the lens. Earlier studies on the binding of 74As-labelled arsenite, arsenate, and DMA to tissues of mice and rabbits in vitro showed that arsenite.was the form which bound most strongly to cellular constituents of the lungs as well as of the liver and kidneys (Vahter and Marafante 1983). However, since there seemed to be no demethylation of DMA in vivo and there was no particular retention of 74As in skin, epithelium of the upper gastrointestinal tract and epididymis as reported for mice exposed to inorganic arsenic (Lindgren et al. 1982), the retention in the tissues of the 74As-DMA exposed mice were probably not a result of binding of inorganic arsenic. Most probably, it is the DMA-metabolite that accumulates in the lungs, intestinal walls, thyroid, and the lens also following exposure to inorganic arsenic. Interestingly, in the lungs of mice and rabbits exposed to inorganic arsenic in the form of arsenite or arsenate, there was a significant higher fraction of the DMA-metabolite and a lower degree of binding of arsenic than in most other tissues (Vahter and Marafante 1983). This would indicate that DMA is taken up more rapidly than inorganic arsenic by the lung ceils or that DMA is retained there longer than inorganic arsenic. Looking at the decrease in tissue concentration of 74As-DMA with time (Figure 3), it is obvious that clearance is slower from the lungs than from the blood, liver, and kidneys. -200

6 264 Marie Vahter et al. Accumulation and long-time retention of arsenic in the thyroid follicles and the lens has been observed in mice also after exposure to inorganic arsenic (Lindgren et al. 1982). In rabbits exposed to 74Asarsenite almost all of the 74As in the thyroid was in the form of DMA (Marafante, unpublished data), indicating that DMA has a higher affinity for the thyroid follicles than inorganic arsenic. Further evidence for this is the fact that in the marmoset monkey, which is unable to methylate inorganic arsenic, there is no specific accumulation of arsenic in the thyroid (Vahter et al. 1982). Possible effects of the DMA accumulation in the thyroid remains to be investigated; this is also true for the long-term retention of DMA in the lens. References Andreae MO (1979) Arsenic speciation in seawater and interstitial waters: The influence of biological-chemical interactions on the chemistry of a trace element. 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