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Metabolism and excretion of orally administrated arsenic trioxide in the hamster
Toxwology, 34 (1985) 113--121
Elsevier ScmntfflC Pubhshers Ireland Ltd
METABOLISM AND EXCRETION OF A R S E N I C T R I O X I D E IN T H E H A M S T E R
ORALLY
ADMINISTRATED
HIROSHI Y A M A U C H I and Y U K I O Y A M A M U R A Department of Publw Health, St Mananna Unwerszty School of Medtcme, 2095, Sugao, M~yamae-ku, Kawasaki 213 (Japan) (Recelved May 15th, 1984) (Accepted October 8th, 1984)
SUMMARY
It was shown that a single dose of arsemc trioxide administered to hamsters was chiefly methylated m vivo into methylarsonic acid (MAA) and dimethylarsinic acid (DMAA), and that inorganic arsenic accounted for the major portion of total arsenic that deposited in organs and tissues, followed by MAA and DMAA in decreasing sequence o f sigmficance. The single oral dose of arsenic trioxide was followed by a very small amount of tnmethylarsenlc compounds (TMA) occurring only m the liver b u t n o t in any other organs, tissues, blood or feces. The distribution pattern of arsemc in the blood followmg the single oral dose of arsenic trioxide was such that inorganic arsenic and MAA occurred chmfly in the blood cells; DMAA, chiefly in the plasma; and the arsenic compounds disappeared rapidly from blood. The single oral dose of arsemc trioxide was further followed by excretion of an amount of arsenic equivalent to a b o u t 60% of the administered dose: 49% in the urine and 11% m the feces. In other words, more arsenic tended to be excreted in the urine. DMAA accounted for the major portion of arsenic excreted in the urine and feces, and this finding re-confirmed that DMAA is the major metabohte of arsenic trioxide. Although it is believed that arsenic trioxide is n o t converted into TMA, the results of the present study suggest that a very small amount of arsenic trioxide is converted into TMA in the liver.
Key words: Inorganic arsenic; Methylarsenic; Dimethylarsenic; Trimethylarsenic; Methylation; Excretion Hlroshl Yamauchi, Department of Public Health, St. Marianna Umverslty School of Medmine, 2095, Sugao, Miyamae-ku, Kawasaki 213, Japan. A b brevlations DMAA, dimethylarsinic acid; In-As, inorganic arsenic; MAA, methylarsonm acid; TMA, tmmethylarsenic compound
Address all correspondence to
0300-483X/85/$03.30 © 1985 Elsevmr Scientific Publishers Ireland Ltd Printed and Published in Ireland
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INTRODUCTION Tnmethylarsenic compounds (TMA) have been detected m seafood [1--3] and h u m a n urine [4--6]. It has also been shown that a dose of dimethylarslnic acid (DMAA) administered to hamsters is partly converted into TMA [7]. On the other hand, results of m a n y experiments on humans [8,9], mice [10], rabbits [11,12] and rats [12,13] indicate that arsemc trioxide is m e t h y l a t e d in vivo into arsemc compounds of low toxicity such as methylarsonic acid (MAA) and DMAA' but n o t m t o TMA. However, there are problems y e t to be solved before we draw a conclusion t h a t arsenm trioxide is n o t converted into TMA; e.g. the precision of an analytic m e t h o d for TMA and the species of experimental animals used. Many reports on the in VlVO distribution of arsenic following administration of arsenm trioxide describe the determlnatmn of total arsenic, while only few deal with the determination of chemmal species of arsenic. There are also papers on the determination of intravisceral arsemc by chemical species m rabbits [11,12] and rats [12,13]. However, the former present data on the chemical species of arsenic in the rabbit liver, kidney and lung, while data from rats (as m the latter) are not always applicable to humans. In other words, there are only a few detailed reports on the observatmns of the chemmal species of arsenic In organs and tissues following adminlstratmn of arsenic trioxide. Also, only a few reports are avmlable on the excretmn ratm of arsenic trioxide in hamster urine and feces. In this study, we followed up the chemical species of arsemc into whmh a single oral dose o f arsenm trioxide given to hamsters would be metabohzed into, those m which it would deposit m organ~ tissues and blood, and the a m o u n t and chemmal species of arsenic excrete~i m u n n e and feces. MATERIALS AND METHODS Male Syrian golden hamsters, weighing 80 +- 7 g, were used. The annnals had free access to a pellet feed manufactured by Japan CLEA, T o k y o , and distilled water. The feed contained 0.32 pg As/g as inorganic arsenic and 0.17 pg As/g as TMA, but was free of either MAA or DMAA. The animals consumed about 8 g of this feed daily, respectively. They were treated with a single oral dose of 4.5 mg/kg body weight of arsenic trioxide (Merck, Germany; 99.95%), which had hrst been dissolved in a very small volume of a solution of NaOH and then diluted with distilled water. They were killed before, and 1, 6, 12, 24, 72 and 120 h after the administration of arsenic trioxide. U n n e and feces samples were collected every 24 h by housmg the hamsters in individual plastic metabolic cages. All materials were preserved frozen at - 2 0 ° C until they were assayed. The hair was washed with distilled water, ethanol and acetone. For the assay arsenic, 0.5--1 g of tissue or feces and 0.5--1 ml of blood or urine were used. These materials were transferred into 25-ml glass-stoppered test tubes, and after the addition of 5 ml o f 2 N NaOH, were heated in a water bath at
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85°C for 3 h. In preliminary experiments, we found that neither MAA, DMAA nor TMA was degraded into any other species of arsenic even when heated at 85°C in 2 N NaOH. Inorganic arsenic, MAA, DMAA and TMA were determined by atomm absorption spectrophotometry [7,14]. The d e t e c t m n limits of the 4 chemical species of arsenm by the m e t h o d were 1 ng, with the coeffmient of varmtmn being not less than 5%, respectively. From the amounts o f morganm arsenic, MAA, DMAA and TMA excreted m the u n n e and feces after the admmistratmn of arsenm t n o m d e , the respective background values described in the Results (mean values before the administration of arsenic trioxide) were deducted. RESULTS Table I shows the concentrations and chemical specms of arsenic m the organs and tissues m the control and the test group following the administration of arsenic trioxide. Only inorganic arsemc was detected in the organs and tissues from the control group. A trace of TMA was, however, detected in the liver. The total arsemc concentrations m the organs and tissues following the administration of arsenic trioxide were greater in the sequence of spleen > liver > lung > skin > kidney > muscle > hair > brain. Inorganm arsenic made up the major portion of the arsenic in the organs and tissues after the administration o f arsemc trioxide, followed by MAA and DMAA. TMA was detected only in the liver. Out of the TMA concentrations in the liver as measured in this study, only those in the samples collected 12 h and 24 h after the admmlstration of arsemc t n o x i d e were significantly high (t-test; P < 0.01), compared to the control group. The methylarsemc compound concentrations m the organs and tissues were such as the MAA concentration was higher than the DMAA concentration m all samples, and DMAA tended to be detected after the appearance of MAA. Inorgamc arsemc disappeared rapidly from the brain, hver, lung and spleen, but its concentrations m the hair, skin, muscle, and kidney remained shghtly high even 120 h after the admimstration of arsemc trioxide, compared to the control group. A trace o f MAA was detected m all organs and tissues assayed, except for the hmr and spleen, even 120 h after the administration of arsenic trioxide, when the arsenic concentration m the kidney was high. DMAA was detected only m the hver 120 h after the admimstratlon of arsenic trioxide. No methylarsemc compounds were found m the hair. Figure 1 depmts changes with time m the arsemc concentration in the blood after the admmlstratmn of arsemc trioxide. The arsenic occurred as 3 chemical specms: inorganic arsenic, MAA and DMAA. No TMA was detected. The inorganm arsemc concentration in the whole blood reached a peak 1 h after, the MAA concentratmn reached a peak 12 h after, and the DMAA concentration reached a peak 24 h after the admimstratmn of arsenic triomde. Inorganm arsenic accounted for the major p o r t m n of the arsenic concentratmn m the whole blood; the MAA concentration was only 1/3 as high as, and the DMAA concentratmn, only 1/10 as high as the inorganic
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TABLE I CONCENTRATIONS Samples
OF ARSENIC COMPOUNDS IN ORGANS AND TISSUES a
Chemical species
Concentration of arsenic (ug As/g wet wt) Hours after administration Cb
Brain
Hair
Kidneys
Liver
Lungs
Muscle
Skin
Spleen
6
12
0 16 0 01 ~.0 01
0 17 < 0 01 --
In-Asc MAA DMAA TMA
0 27 . . .
0 27 . . .
0 22 . . .
0 19 . . .
0 25 . . .
. . .
0 20
0.65 0.06 -.
O 66 0 20 0 04 .
0 56 0.36 0.09 .
0 35 0 51 0 13 .
0 20 0 50 -.
3 24 0 04 <0.01 <001 <001
1 55 0 16 <0 01 <0.01
1 53 0 20 0.02 001
0 72 0 16 0.08 004
0 34 0 03 0 02 <001
0 18 <0.01 <0 01 -.001
In-Asc MAA DMAA TMAA
0 36 1 11 -<0 01 --. .
1.17 0.17 0.01 .
1 11 0 49 0.02 .
0.86 0 44 0 13 .
0 41 0 01 0 02
0 37 <0 01 -.
In-As c MAA DMAA TMA
0 24 --.
0 20 --.
0 36 <0.01 <0 01 .
0.49 0.03 <0 01 .
0 37 0.09 0.02 .
0.36 0 03 <0 01 .
0 36 0 01
In-Asc MAA DMAA TMA
0 13 --.
0.26 --.
0.61 0.02 <0 01 .
0 95 0.08 <0 01 .
O 96 0 II 0 02 .
0 60 0 03 0 02
1 14
2 33 <0 01 -.
3.59 0 24 -.
2.46 0 52 <0 01 .
2 53 0 40 <0 01 .
In-Asc MAA DMAA TMA
In-Asc MAA DMAA TMA
-
-
-. 0 15 --
-
-
-
-.
.
0 22 0 02 0 02
120
0 17 --.
.
0.21 0.01 0 01 .
72
0 17 --.
In-Asc MAA DMAA TMA
0 17 < 0 01 --
24
In-Asc MAA DMAA TMA
aMean for 5 hamsters. bc, controls. CIn-As, m o r g a m c a r s e n i c --, n o t detected.
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1
.
. 0 29
0 36 . . .
.
0 17 0 17 -.
-.
.
0 42 <0 01 -.
1 99 0 03 -.
--.
1 35
12 • In-As O MAA • DMAA
Wo%
:~08 "0 o 0
~
PLASMA
06
u 04
g ~o2 O
1,1
controls
1
24
72 Hours
120 c after
1
24
72
120
administration
Fig 1 Coneentratmns of arsenic compounds in whole blood and plasma of hamsters following a single oral admlnmtratmn of arsenic trioxlde M e a n + S D of 5 hamsters.
arsenic concentration. The distribution pattern of arsenic in the blood was such as inorganic arsenic and MAA occurred chiefly in the blood cells, and DMAA, chiefly in the plasma. The arsenic disappeared rapidly from the whole blood and the plasma, with the arsenic concentrations in them havmg dropped to about the control values 72 h after the administration of ajsenlc trioxide. Table II shows that excretion pattern of arsenic in the urine following the TABLE
II
URINARY ARSENIC COMPOUNDS EXCRETION ADMINISTRATION OF ARSENIC TRIOXIDE a
FOLLOWING
A SINGLE ORAL
Day a f t e r admmmtrat]on
A m o u n t s o f arsemc (ug A s / d a y ) b In-As c
MMA
DMAA
TMA
1 2 3 4 5 Total %d
682 +273 1 2 6 +_ 5 9 0 0.87 + 0.60 0.37+- 0 3 4 0 6 8 +_ 0 80 8 2 8 +-27 1 23 3
4 72+_2.91 3.60+-2.09 0.27 + 0 30 0.21+011 0.19 +_ 0.14 8.99+-4.56 2 5
11.7 + 9 . 0 2 52.2 +_4.02 7.85 _+ 1.98 3.90+-039 2.59 +- 1.08 78.3 +-5.87 22.1
0.40+_0.37 0.93+_049 0.34 _+ 0.17 0.43+-0.11 0 . 3 9 +- 0.10 2.49+-2.20 0.7
Total 85.0 + 2 1 7 69.3 _+ 6.70 9.33 +_ 1.94 4 9 1 _ + 0.95 3.85 +_ 2.55 172 +-24.2 48 5
a M e a n +- S,D. for 5 h a m s t e r s b A f t e r c o r r e c h o n for a basal dally e x c r e t i o n o f 1.69 -+ 0.69 #g A s / d a y (In-As 43%, M A A 2%, D M A A 12%, T M A 43%) CIn-As, inorganic arsemc d p e r c e n t a g e o f a d m i n i s t e r e d dose e x c r e t e d m urine ( m e a n dose o f 3 5 5 / ~ g As)
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TABLE III FECAL ARSENIC COMPOUNDS EXCRETION FOLLOWING A SINGLE ORAL ADMINISTRATION OF ARSENIC TRIOXIDEa Days after admmmtration 1 2 3 4 5 Total %d
Amounts of arsemc (ug As/day)b In-As c
MAA
DMAA
TMA
Total
523"+283 13 7 _+513 1.01 + 0 5 4 055-+0.22 0 59 -+ 0 24 21 1 -+367 59
199"+093 773-+462 073-+029 017-+007 0 15 -+ 0 05 108 _4-213 30
031"+011 447"+189 162-+1.99 0.13-+007 0 21 -+ 0.15 6 74-+268 19
-------
752"+387 260 _+647 349_+242 089-+024 0.90 + 0 51 3 8 8 4-4 97 10.9
aMean -+ S D for 5 hamsters bAfter correctmn for a basal daffy excretmn of 0.42 -+ 0 16 ug As/day (In-As 93%, MAA 5%, DMAA 2%) CIn-As, morganm arsenm. dpercentage of adminmtered dose excreted in feces (mean dose of 355 ug As)
a d m i n i s t r a t i o n o f arsenic trioxide. T h e m e a n e x c r e t m n o f a r s e m c ,n the urine d u r m g t h e 3 d a y s b e f o r e t h e a d m i m s t r a t i o n o f arsenic t r i o x i d e was 1.69 + 0 . 6 9 pg A s / d a y ( m a d e u p o f 43% m o r g a n i c arsenic, 2% M A A , 12% D M A A a n d 43% TMA). T h e t o t a l arsemc e x c r e t i o n d u n n g t h e first 24 h after t h e a d m i m s t r a t l o n o f arsenm t r i o x i d e was 85.0 + 21.7 #g A s / d a y , w h m h c o r r e s p o n d e d t o 23.9% o f the a d m m m t e r e d dose o f arsenic. A b o u t 50% o f t h e a d m i n i s t e r e d dose o f arsenm was e x c r e t e d d u r i n g t h e 120 h a f t e r t h e a d m m m t r a t l o n o f arsenm trioxide. I n o r g a n m arsenm a c c o u n t e d f o r a b o u t o n e half o f the t o t a l arsenm e x c r e t i o n m the u n n e . O n the o t h e r h a n d , D M A A a c c o u n t e d f o r the m a j o r p o r t i o n o f t h e m e t h y l a r s e n m c o m p o u n d s e x c r e t e d , f o l l o w e d b y M A A a n d b y a v e r y small a m o u n t o f TMA. Table I I I s h o w s the e x c r e t i o n p a t t e r n o f arsenm in t h e feces after t h e a d m i n i s t r a t i o n o f arsenic trioxide. T h e m e a n arsenic e x c r e t i o n m t h e feces d u r i n g t h e 3 d a y s b e f o r e t h e a d m i n i s t r a t i o n o f arsemc t r i o x i d e was 0.42 + 0.16 ~g A s / d a y ( m a d e u p o f 93% i n o r g a m c arsemc, 5% M A A a n d 2% D M A A ) . T h e t o t a l arsemc e x c r e t i o n d u r i n g t h e first 24 h a f t e r t h e a d m i n l s t r a t m n o f arsenic t r i o x i d e was 7.52 + 3.87 # g A s / d a y , w h i c h c o r r e s p o n d e d t o 2.1% o f t h e a d m i n i s t e r e d dose o f arsenic. A b o u t 11% o f t h e a d m i m s t e r e d dose o f arsenic was e x c r e t e d d u r i n g t h e 120 h after t h e a d m i m s t r a t m n o f arsenic t r i o x i d e . I n o r g a n m arsenic a c c o u n t e d f o r t h e greatest p a r t o f t h e arsemc d e t e c t e d m t h e feces, f o l l o w e d b y M A A a n d D M A A . N o T M A was d e t e c t e d m t h e feces. In o t h e r w o r d s , w h e n hamsters were given a single oral dose o f arsenm t r m x i d e , 59.4% o f t h e a d m i n i s t e r e d a m o u n t o f arsemc was e x c r e t e d d u n n g t h e 120 h a f t e r t h e a d m i m s t r a t i o n , a n d st t e n d e d t o be m o r e e x c r e t e d in t h e u n n e . A b o u t 1/2 o f t h e t o t a l arsenic e x c r e t e d m t h e u n n e a n d feces
118
occurred as inorganic arsemc. DMAA made up the greatest part of the methylarsenic compounds excreted, accounting for about 24% of the administered a m o u n t of arsenic: DMAA was thus shown to be the major metabolite of arsenm trioxide. DISCUSSION The finding m this study that arsenm triomde was converted in vivo into MAA and DMAA was consistent with the results of experiments on humans [8,9] and animals [10--13]. It is beheved that arsenic trioxide is not converted into TMA m mammalians. The amounts of TMA detected in the liver 12 h and 24 h after the smgle oral dose of arsenic trioxide m this study differed significantly from the control values, suggesting that a very small a m o u n t of arsenic t n o m d e would be converted into TMA m the liver. In other words, it appears too hasty to conclude that arsenic trioxide is not converted into TMA in the mammahans. On the other hand, it is true that arsemc trioxide is only httle converted into TMA. An a m o u n t of TMA equivalent to 0.7% of the admimstered dose of arsenic was excreted m the unne, but this excretion did not differ signifmantly from the control value. This statistmally insignificant excretion of TMA m the urine may be t h o u g h t to result from the in vivo formation of TMA in only very small amounts and from the TMA in the feed masking the TMA probably formed in vivo, causing confusions. Vahter [10] described that the conversion of arsenm trioxide into DMAA was dose~lependent. In a previous study [ 7 ], we observed that a single oral dose of DMAA administered to hamsters was followed by conversion of about 16% of the dose into TMA. In the present study, about 25% of the admimstered dose of arsenic trioxide was converted into DMAA, and from this finding, it may be estimated t h a t only a small a m o u n t of DMAA is retained m hamsters. The findings m the aforementmned previous study by the present authors and the present study appear to indicate that the conversion of DMAA to TMA is dose-dependent, but it is not clear to us at present whether this is true or not. The arsenic compounds in the organs and tissues after the admimstration of arsenic triomde showed the following tendency: inorganic arsenm accounted for about 90% or more of the total arsenm in the early stages, Le., 1 h and 6 h, after the administration of arsenic trioxide; the ratio of MAA increased with time, the increase being marked in the kidney and lung in partmular; DMAA also increased m parallel with MAA, but accounted for only less than about 10% of the total arsemc concentratmn. In experiments on rabbits [11,12] and rats [12,13], inorganic arsenm tended to account for a large portion of arsenic within 6 h after administration of arsenic tnoxide, and the fmdmgs m this study on hamsters agree with these findings. However, the d i s t n b u t m n pattern of methylarsenm compounds was different in these studms: DMAA accounted for a large part in rabbits [ 11,12 ] and rats [ 12,13 ], while MAA made up a large portion in hamsters. On the other hand, the excretmn of a large a m o u n t of DMAA in the urine and feces, accounting for
119
about 24% of the admimstered a m o u n t of arsenic, suggested t h a t a large a m o u n t of DMAA might be produced in vivo but might not be accumulated in the organs and tissues. The total arsenic a m o u n t excreted m the u n n e and feces during the 5 days after the administration of arsenic trmxide corresponded to about 60% of the total a m o u n t of arsenic administered. Comparison between the arsenm excretion rate during the first 5 days after the administration of arsenic trioxide to hamsters and that after the administration of the same c o m p o u n d to rats [13] showed t h a t the former was about 3 times as high as the latter. The degree of affinity of the blood for arsemc seemed responsible for the difference in the excretion of arsenic outside the b o d y between rats and hamsters. Because the rat red blood cells are very affinitlve for arsenic, it is "retained m the rat body for a long time. On the other hand, lnorgamc arsenic and MAA were shown to disappear rapidly from the hamster blood, whmh seemed contributory to the high excretion rate o f arsenic. The total excretion o f arsenic trioxide in experimental animals of other species than the rat is such as 85.3% [12] and 92.6% [11] in rabbits, and 75.4--91.2% [10] m mice. In other words, the excretion of arsenic trioxide m hamsters tends to be low, compared to rabbits and mice. In humans [ 8 ] , the mean u n n a r y excretion rate of arsenic during the 3 days after administration of arsenic trioxide was shown to be 62% of the administered a m o u n t of arsenic, and Buchet et al. [9] reported that the excretion rate of arsenic d u n n g the 4 days after the oral administration of arsenm trioxide was 45.1% or 48.8%. In this study, the total arsenic excretion rate m the u n n e d u n n g the 5 days after the administratmn of arsenic trioxide was 48.5%, which was in close agreement with the findings in humans [8,9]. Therefore, we are of the opinion that the data from hamsters may be extrapolated to the data from humans, compared to the data from rats, mice and rabbits. ACKNOWLEDGEMENT
This study was aided in part by the Grant in Scientific Research from the Ministry of Education, Japan (1983). REFERENCES 1 J.S E d m o n d s and K A Francescom, Methylated arsemc from marine fauna. Nature, 265 (1977) 436 2 H Yamauchi and Y. Yamamura, Arsemte, arsenate and methylarsenm in raw foods Jpn J Pubhc Health, 27 (1980) 647. 3 H N o r m and A. Chrmtakopoulos, Evidence for the presence of arsenobetame and another organo arsenmal m shrimps Chemosphere, 11 (1982) 287. 4 J.R Cannon, J S Edmonds, K A. Francesconi, C.L. Raston, J.S. Saunders, B.W Skelton and A H White, Isolation, crystal structure and synthesis of arseno-betame, a constituent of the western rock lobster, the dusky shark, and some samples of h u m a n urine. Aust. J. Chem., 34 (1981) 787. 5 J B. Luten, G Riekwel-Booy and A. Rauchbaar, Occurrence of arsenic m plame
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(Pleuronectes platessa), nature of organo-arsenm compound present and its excretion by man. Environ. Health Perspect., 45 (1982) 165. 6 H. Yamauchi and Y. Yamamura, Metabolism and excretion of orally ingested trimethylarsenic in man Bull. Environ. Contam. Toxmol., 32 (1984) 682. 7 H Yamauchl and Y Yamamura, Metabolism and excretion of orally admmLstered dlmethylarslnlC acid m the hamster Toxmol. Appl. Phv.rmacol, 74 (1984) 134. 8 H Yamauchi and Y. Yamamura, Dynamic change of inorganm arsenm and methylarsenm compounds in human urine after oral intake as arsenic tmoxlde. Ind. Health, 17 (1979) 79 9 J P Buchet, R. Lauwerys and H Roels, Comparison of the urinary excretion of arsenic metabolites after a single oral dose of sodium arsenlte, monomethylarsonate, or dlmethylarsinate m man Int. Arch. Occup Environ Health, 48 (1981) 71 10 M. Vahter, Blotransformation of trivalent and pentavalent morgamc arsenm in mine and rats. Environ. R e s , 25 (1981) 286 11 F Bertolero, E. Marafante, J. Edel Rade, R Pietra and E. Sabblom, Blotransformatlon and lntracellular binding of arsenic m tissues of rabbits after intraperitoneal admmmtratlon of 74As labelled arsemte. Toxicology, 20 (1981) 35 12 E Marafante, F Bertolero, J Edel, R. Pietra and E. Sabbloni, Intracellular interaction and biotransformatlon of arsenite m rats and rabbits Sci Total Environ, 24 (1982) 27 13 H. Yamauchl, M. Iwata and Y. Yamamura, Matabolism and excretion of arsenic tmoxlde in rats. Jpn J Ind. Health, 22 (1980) 111 14 H. Yamauchl and Y. Yamamura, Urinary inorganic arsemc and methylarsemc excretion following arsenate-rich seaweed lngestmn Jp n J Ind Health, 21 (1979) 47
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Elsevier ScmntfflC Pubhshers Ireland Ltd
METABOLISM AND EXCRETION OF A R S E N I C T R I O X I D E IN T H E H A M S T E R
ORALLY
ADMINISTRATED
HIROSHI Y A M A U C H I and Y U K I O Y A M A M U R A Department of Publw Health, St Mananna Unwerszty School of Medtcme, 2095, Sugao, M~yamae-ku, Kawasaki 213 (Japan) (Recelved May 15th, 1984) (Accepted October 8th, 1984)
SUMMARY
It was shown that a single dose of arsemc trioxide administered to hamsters was chiefly methylated m vivo into methylarsonic acid (MAA) and dimethylarsinic acid (DMAA), and that inorganic arsenic accounted for the major portion of total arsenic that deposited in organs and tissues, followed by MAA and DMAA in decreasing sequence o f sigmficance. The single oral dose of arsenic trioxide was followed by a very small amount of tnmethylarsenlc compounds (TMA) occurring only m the liver b u t n o t in any other organs, tissues, blood or feces. The distribution pattern of arsemc in the blood followmg the single oral dose of arsenic trioxide was such that inorganic arsenic and MAA occurred chmfly in the blood cells; DMAA, chiefly in the plasma; and the arsenic compounds disappeared rapidly from blood. The single oral dose of arsemc trioxide was further followed by excretion of an amount of arsenic equivalent to a b o u t 60% of the administered dose: 49% in the urine and 11% m the feces. In other words, more arsenic tended to be excreted in the urine. DMAA accounted for the major portion of arsenic excreted in the urine and feces, and this finding re-confirmed that DMAA is the major metabohte of arsenic trioxide. Although it is believed that arsenic trioxide is n o t converted into TMA, the results of the present study suggest that a very small amount of arsenic trioxide is converted into TMA in the liver.
Key words: Inorganic arsenic; Methylarsenic; Dimethylarsenic; Trimethylarsenic; Methylation; Excretion Hlroshl Yamauchi, Department of Public Health, St. Marianna Umverslty School of Medmine, 2095, Sugao, Miyamae-ku, Kawasaki 213, Japan. A b brevlations DMAA, dimethylarsinic acid; In-As, inorganic arsenic; MAA, methylarsonm acid; TMA, tmmethylarsenic compound
Address all correspondence to
0300-483X/85/$03.30 © 1985 Elsevmr Scientific Publishers Ireland Ltd Printed and Published in Ireland
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INTRODUCTION Tnmethylarsenic compounds (TMA) have been detected m seafood [1--3] and h u m a n urine [4--6]. It has also been shown that a dose of dimethylarslnic acid (DMAA) administered to hamsters is partly converted into TMA [7]. On the other hand, results of m a n y experiments on humans [8,9], mice [10], rabbits [11,12] and rats [12,13] indicate that arsemc trioxide is m e t h y l a t e d in vivo into arsemc compounds of low toxicity such as methylarsonic acid (MAA) and DMAA' but n o t m t o TMA. However, there are problems y e t to be solved before we draw a conclusion t h a t arsenm trioxide is n o t converted into TMA; e.g. the precision of an analytic m e t h o d for TMA and the species of experimental animals used. Many reports on the in VlVO distribution of arsenic following administration of arsenm trioxide describe the determlnatmn of total arsenic, while only few deal with the determination of chemmal species of arsenic. There are also papers on the determination of intravisceral arsemc by chemical species m rabbits [11,12] and rats [12,13]. However, the former present data on the chemical species of arsenic in the rabbit liver, kidney and lung, while data from rats (as m the latter) are not always applicable to humans. In other words, there are only a few detailed reports on the observatmns of the chemmal species of arsenic In organs and tissues following adminlstratmn of arsenic trioxide. Also, only a few reports are avmlable on the excretmn ratm of arsenic trioxide in hamster urine and feces. In this study, we followed up the chemical species of arsemc into whmh a single oral dose o f arsenm trioxide given to hamsters would be metabohzed into, those m which it would deposit m organ~ tissues and blood, and the a m o u n t and chemmal species of arsenic excrete~i m u n n e and feces. MATERIALS AND METHODS Male Syrian golden hamsters, weighing 80 +- 7 g, were used. The annnals had free access to a pellet feed manufactured by Japan CLEA, T o k y o , and distilled water. The feed contained 0.32 pg As/g as inorganic arsenic and 0.17 pg As/g as TMA, but was free of either MAA or DMAA. The animals consumed about 8 g of this feed daily, respectively. They were treated with a single oral dose of 4.5 mg/kg body weight of arsenic trioxide (Merck, Germany; 99.95%), which had hrst been dissolved in a very small volume of a solution of NaOH and then diluted with distilled water. They were killed before, and 1, 6, 12, 24, 72 and 120 h after the administration of arsenic trioxide. U n n e and feces samples were collected every 24 h by housmg the hamsters in individual plastic metabolic cages. All materials were preserved frozen at - 2 0 ° C until they were assayed. The hair was washed with distilled water, ethanol and acetone. For the assay arsenic, 0.5--1 g of tissue or feces and 0.5--1 ml of blood or urine were used. These materials were transferred into 25-ml glass-stoppered test tubes, and after the addition of 5 ml o f 2 N NaOH, were heated in a water bath at
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85°C for 3 h. In preliminary experiments, we found that neither MAA, DMAA nor TMA was degraded into any other species of arsenic even when heated at 85°C in 2 N NaOH. Inorganic arsenic, MAA, DMAA and TMA were determined by atomm absorption spectrophotometry [7,14]. The d e t e c t m n limits of the 4 chemical species of arsenm by the m e t h o d were 1 ng, with the coeffmient of varmtmn being not less than 5%, respectively. From the amounts o f morganm arsenic, MAA, DMAA and TMA excreted m the u n n e and feces after the admmistratmn of arsenm t n o m d e , the respective background values described in the Results (mean values before the administration of arsenic trioxide) were deducted. RESULTS Table I shows the concentrations and chemical specms of arsenic m the organs and tissues m the control and the test group following the administration of arsenic trioxide. Only inorganic arsemc was detected in the organs and tissues from the control group. A trace of TMA was, however, detected in the liver. The total arsemc concentrations m the organs and tissues following the administration of arsenic trioxide were greater in the sequence of spleen > liver > lung > skin > kidney > muscle > hair > brain. Inorganm arsenic made up the major portion of the arsenic in the organs and tissues after the administration o f arsemc trioxide, followed by MAA and DMAA. TMA was detected only in the liver. Out of the TMA concentrations in the liver as measured in this study, only those in the samples collected 12 h and 24 h after the admmlstration of arsemc t n o x i d e were significantly high (t-test; P < 0.01), compared to the control group. The methylarsemc compound concentrations m the organs and tissues were such as the MAA concentration was higher than the DMAA concentration m all samples, and DMAA tended to be detected after the appearance of MAA. Inorgamc arsemc disappeared rapidly from the brain, hver, lung and spleen, but its concentrations m the hair, skin, muscle, and kidney remained shghtly high even 120 h after the admimstration of arsemc trioxide, compared to the control group. A trace o f MAA was detected m all organs and tissues assayed, except for the hmr and spleen, even 120 h after the administration of arsenic trioxide, when the arsenic concentration m the kidney was high. DMAA was detected only m the hver 120 h after the admimstratlon of arsenic trioxide. No methylarsemc compounds were found m the hair. Figure 1 depmts changes with time m the arsemc concentration in the blood after the admmlstratmn of arsemc trioxide. The arsenic occurred as 3 chemical specms: inorganic arsenic, MAA and DMAA. No TMA was detected. The inorganm arsemc concentration in the whole blood reached a peak 1 h after, the MAA concentratmn reached a peak 12 h after, and the DMAA concentration reached a peak 24 h after the admimstratmn of arsenic triomde. Inorganm arsenic accounted for the major p o r t m n of the arsenic concentratmn m the whole blood; the MAA concentration was only 1/3 as high as, and the DMAA concentratmn, only 1/10 as high as the inorganic
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TABLE I CONCENTRATIONS Samples
OF ARSENIC COMPOUNDS IN ORGANS AND TISSUES a
Chemical species
Concentration of arsenic (ug As/g wet wt) Hours after administration Cb
Brain
Hair
Kidneys
Liver
Lungs
Muscle
Skin
Spleen
6
12
0 16 0 01 ~.0 01
0 17 < 0 01 --
In-Asc MAA DMAA TMA
0 27 . . .
0 27 . . .
0 22 . . .
0 19 . . .
0 25 . . .
. . .
0 20
0.65 0.06 -.
O 66 0 20 0 04 .
0 56 0.36 0.09 .
0 35 0 51 0 13 .
0 20 0 50 -.
3 24 0 04 <0.01 <001 <001
1 55 0 16 <0 01 <0.01
1 53 0 20 0.02 001
0 72 0 16 0.08 004
0 34 0 03 0 02 <001
0 18 <0.01 <0 01 -.001
In-Asc MAA DMAA TMAA
0 36 1 11 -<0 01 --. .
1.17 0.17 0.01 .
1 11 0 49 0.02 .
0.86 0 44 0 13 .
0 41 0 01 0 02
0 37 <0 01 -.
In-As c MAA DMAA TMA
0 24 --.
0 20 --.
0 36 <0.01 <0 01 .
0.49 0.03 <0 01 .
0 37 0.09 0.02 .
0.36 0 03 <0 01 .
0 36 0 01
In-Asc MAA DMAA TMA
0 13 --.
0.26 --.
0.61 0.02 <0 01 .
0 95 0.08 <0 01 .
O 96 0 II 0 02 .
0 60 0 03 0 02
1 14
2 33 <0 01 -.
3.59 0 24 -.
2.46 0 52 <0 01 .
2 53 0 40 <0 01 .
In-Asc MAA DMAA TMA
In-Asc MAA DMAA TMA
-
-
-. 0 15 --
-
-
-
-.
.
0 22 0 02 0 02
120
0 17 --.
.
0.21 0.01 0 01 .
72
0 17 --.
In-Asc MAA DMAA TMA
0 17 < 0 01 --
24
In-Asc MAA DMAA TMA
aMean for 5 hamsters. bc, controls. CIn-As, m o r g a m c a r s e n i c --, n o t detected.
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1
.
. 0 29
0 36 . . .
.
0 17 0 17 -.
-.
.
0 42 <0 01 -.
1 99 0 03 -.
--.
1 35
12 • In-As O MAA • DMAA
Wo%
:~08 "0 o 0
~
PLASMA
06
u 04
g ~o2 O
1,1
controls
1
24
72 Hours
120 c after
1
24
72
120
administration
Fig 1 Coneentratmns of arsenic compounds in whole blood and plasma of hamsters following a single oral admlnmtratmn of arsenic trioxlde M e a n + S D of 5 hamsters.
arsenic concentration. The distribution pattern of arsenic in the blood was such as inorganic arsenic and MAA occurred chiefly in the blood cells, and DMAA, chiefly in the plasma. The arsenic disappeared rapidly from the whole blood and the plasma, with the arsenic concentrations in them havmg dropped to about the control values 72 h after the administration of ajsenlc trioxide. Table II shows that excretion pattern of arsenic in the urine following the TABLE
II
URINARY ARSENIC COMPOUNDS EXCRETION ADMINISTRATION OF ARSENIC TRIOXIDE a
FOLLOWING
A SINGLE ORAL
Day a f t e r admmmtrat]on
A m o u n t s o f arsemc (ug A s / d a y ) b In-As c
MMA
DMAA
TMA
1 2 3 4 5 Total %d
682 +273 1 2 6 +_ 5 9 0 0.87 + 0.60 0.37+- 0 3 4 0 6 8 +_ 0 80 8 2 8 +-27 1 23 3
4 72+_2.91 3.60+-2.09 0.27 + 0 30 0.21+011 0.19 +_ 0.14 8.99+-4.56 2 5
11.7 + 9 . 0 2 52.2 +_4.02 7.85 _+ 1.98 3.90+-039 2.59 +- 1.08 78.3 +-5.87 22.1
0.40+_0.37 0.93+_049 0.34 _+ 0.17 0.43+-0.11 0 . 3 9 +- 0.10 2.49+-2.20 0.7
Total 85.0 + 2 1 7 69.3 _+ 6.70 9.33 +_ 1.94 4 9 1 _ + 0.95 3.85 +_ 2.55 172 +-24.2 48 5
a M e a n +- S,D. for 5 h a m s t e r s b A f t e r c o r r e c h o n for a basal dally e x c r e t i o n o f 1.69 -+ 0.69 #g A s / d a y (In-As 43%, M A A 2%, D M A A 12%, T M A 43%) CIn-As, inorganic arsemc d p e r c e n t a g e o f a d m i n i s t e r e d dose e x c r e t e d m urine ( m e a n dose o f 3 5 5 / ~ g As)
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TABLE III FECAL ARSENIC COMPOUNDS EXCRETION FOLLOWING A SINGLE ORAL ADMINISTRATION OF ARSENIC TRIOXIDEa Days after admmmtration 1 2 3 4 5 Total %d
Amounts of arsemc (ug As/day)b In-As c
MAA
DMAA
TMA
Total
523"+283 13 7 _+513 1.01 + 0 5 4 055-+0.22 0 59 -+ 0 24 21 1 -+367 59
199"+093 773-+462 073-+029 017-+007 0 15 -+ 0 05 108 _4-213 30
031"+011 447"+189 162-+1.99 0.13-+007 0 21 -+ 0.15 6 74-+268 19
-------
752"+387 260 _+647 349_+242 089-+024 0.90 + 0 51 3 8 8 4-4 97 10.9
aMean -+ S D for 5 hamsters bAfter correctmn for a basal daffy excretmn of 0.42 -+ 0 16 ug As/day (In-As 93%, MAA 5%, DMAA 2%) CIn-As, morganm arsenm. dpercentage of adminmtered dose excreted in feces (mean dose of 355 ug As)
a d m i n i s t r a t i o n o f arsenic trioxide. T h e m e a n e x c r e t m n o f a r s e m c ,n the urine d u r m g t h e 3 d a y s b e f o r e t h e a d m i m s t r a t i o n o f arsenic t r i o x i d e was 1.69 + 0 . 6 9 pg A s / d a y ( m a d e u p o f 43% m o r g a n i c arsenic, 2% M A A , 12% D M A A a n d 43% TMA). T h e t o t a l arsemc e x c r e t i o n d u n n g t h e first 24 h after t h e a d m i m s t r a t l o n o f arsenm t r i o x i d e was 85.0 + 21.7 #g A s / d a y , w h m h c o r r e s p o n d e d t o 23.9% o f the a d m m m t e r e d dose o f arsenic. A b o u t 50% o f t h e a d m i n i s t e r e d dose o f arsenm was e x c r e t e d d u r i n g t h e 120 h a f t e r t h e a d m m m t r a t l o n o f arsenm trioxide. I n o r g a n m arsenm a c c o u n t e d f o r a b o u t o n e half o f the t o t a l arsenm e x c r e t i o n m the u n n e . O n the o t h e r h a n d , D M A A a c c o u n t e d f o r the m a j o r p o r t i o n o f t h e m e t h y l a r s e n m c o m p o u n d s e x c r e t e d , f o l l o w e d b y M A A a n d b y a v e r y small a m o u n t o f TMA. Table I I I s h o w s the e x c r e t i o n p a t t e r n o f arsenm in t h e feces after t h e a d m i n i s t r a t i o n o f arsenic trioxide. T h e m e a n arsenic e x c r e t i o n m t h e feces d u r i n g t h e 3 d a y s b e f o r e t h e a d m i n i s t r a t i o n o f arsemc t r i o x i d e was 0.42 + 0.16 ~g A s / d a y ( m a d e u p o f 93% i n o r g a m c arsemc, 5% M A A a n d 2% D M A A ) . T h e t o t a l arsemc e x c r e t i o n d u r i n g t h e first 24 h a f t e r t h e a d m i n l s t r a t m n o f arsenic t r i o x i d e was 7.52 + 3.87 # g A s / d a y , w h i c h c o r r e s p o n d e d t o 2.1% o f t h e a d m i n i s t e r e d dose o f arsenic. A b o u t 11% o f t h e a d m i m s t e r e d dose o f arsenic was e x c r e t e d d u r i n g t h e 120 h after t h e a d m i m s t r a t m n o f arsenic t r i o x i d e . I n o r g a n m arsenic a c c o u n t e d f o r t h e greatest p a r t o f t h e arsemc d e t e c t e d m t h e feces, f o l l o w e d b y M A A a n d D M A A . N o T M A was d e t e c t e d m t h e feces. In o t h e r w o r d s , w h e n hamsters were given a single oral dose o f arsenm t r m x i d e , 59.4% o f t h e a d m i n i s t e r e d a m o u n t o f arsemc was e x c r e t e d d u n n g t h e 120 h a f t e r t h e a d m i m s t r a t i o n , a n d st t e n d e d t o be m o r e e x c r e t e d in t h e u n n e . A b o u t 1/2 o f t h e t o t a l arsenic e x c r e t e d m t h e u n n e a n d feces
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occurred as inorganic arsemc. DMAA made up the greatest part of the methylarsenic compounds excreted, accounting for about 24% of the administered a m o u n t of arsenic: DMAA was thus shown to be the major metabolite of arsenm trioxide. DISCUSSION The finding m this study that arsenm triomde was converted in vivo into MAA and DMAA was consistent with the results of experiments on humans [8,9] and animals [10--13]. It is beheved that arsenic trioxide is not converted into TMA m mammalians. The amounts of TMA detected in the liver 12 h and 24 h after the smgle oral dose of arsenic trioxide m this study differed significantly from the control values, suggesting that a very small a m o u n t of arsenic t n o m d e would be converted into TMA m the liver. In other words, it appears too hasty to conclude that arsenic trioxide is not converted into TMA in the mammahans. On the other hand, it is true that arsemc trioxide is only httle converted into TMA. An a m o u n t of TMA equivalent to 0.7% of the admimstered dose of arsenic was excreted m the unne, but this excretion did not differ signifmantly from the control value. This statistmally insignificant excretion of TMA m the urine may be t h o u g h t to result from the in vivo formation of TMA in only very small amounts and from the TMA in the feed masking the TMA probably formed in vivo, causing confusions. Vahter [10] described that the conversion of arsenm trioxide into DMAA was dose~lependent. In a previous study [ 7 ], we observed that a single oral dose of DMAA administered to hamsters was followed by conversion of about 16% of the dose into TMA. In the present study, about 25% of the admimstered dose of arsenic trioxide was converted into DMAA, and from this finding, it may be estimated t h a t only a small a m o u n t of DMAA is retained m hamsters. The findings m the aforementmned previous study by the present authors and the present study appear to indicate that the conversion of DMAA to TMA is dose-dependent, but it is not clear to us at present whether this is true or not. The arsenic compounds in the organs and tissues after the admimstration of arsenic triomde showed the following tendency: inorganic arsenm accounted for about 90% or more of the total arsenm in the early stages, Le., 1 h and 6 h, after the administration of arsenic trioxide; the ratio of MAA increased with time, the increase being marked in the kidney and lung in partmular; DMAA also increased m parallel with MAA, but accounted for only less than about 10% of the total arsemc concentratmn. In experiments on rabbits [11,12] and rats [12,13], inorganic arsenm tended to account for a large portion of arsenic within 6 h after administration of arsenic tnoxide, and the fmdmgs m this study on hamsters agree with these findings. However, the d i s t n b u t m n pattern of methylarsenm compounds was different in these studms: DMAA accounted for a large part in rabbits [ 11,12 ] and rats [ 12,13 ], while MAA made up a large portion in hamsters. On the other hand, the excretmn of a large a m o u n t of DMAA in the urine and feces, accounting for
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about 24% of the admimstered a m o u n t of arsenic, suggested t h a t a large a m o u n t of DMAA might be produced in vivo but might not be accumulated in the organs and tissues. The total arsenic a m o u n t excreted m the u n n e and feces during the 5 days after the administration of arsenic trmxide corresponded to about 60% of the total a m o u n t of arsenic administered. Comparison between the arsenm excretion rate during the first 5 days after the administration of arsenic trioxide to hamsters and that after the administration of the same c o m p o u n d to rats [13] showed t h a t the former was about 3 times as high as the latter. The degree of affinity of the blood for arsemc seemed responsible for the difference in the excretion of arsenic outside the b o d y between rats and hamsters. Because the rat red blood cells are very affinitlve for arsenic, it is "retained m the rat body for a long time. On the other hand, lnorgamc arsenic and MAA were shown to disappear rapidly from the hamster blood, whmh seemed contributory to the high excretion rate o f arsenic. The total excretion o f arsenic trioxide in experimental animals of other species than the rat is such as 85.3% [12] and 92.6% [11] in rabbits, and 75.4--91.2% [10] m mice. In other words, the excretion of arsenic trioxide m hamsters tends to be low, compared to rabbits and mice. In humans [ 8 ] , the mean u n n a r y excretion rate of arsenic during the 3 days after administration of arsenic trioxide was shown to be 62% of the administered a m o u n t of arsenic, and Buchet et al. [9] reported that the excretion rate of arsenic d u n n g the 4 days after the oral administration of arsenm trioxide was 45.1% or 48.8%. In this study, the total arsenic excretion rate m the u n n e d u n n g the 5 days after the administratmn of arsenic trioxide was 48.5%, which was in close agreement with the findings in humans [8,9]. Therefore, we are of the opinion that the data from hamsters may be extrapolated to the data from humans, compared to the data from rats, mice and rabbits. ACKNOWLEDGEMENT
This study was aided in part by the Grant in Scientific Research from the Ministry of Education, Japan (1983). REFERENCES 1 J.S E d m o n d s and K A Francescom, Methylated arsemc from marine fauna. Nature, 265 (1977) 436 2 H Yamauchi and Y. Yamamura, Arsemte, arsenate and methylarsenm in raw foods Jpn J Pubhc Health, 27 (1980) 647. 3 H N o r m and A. Chrmtakopoulos, Evidence for the presence of arsenobetame and another organo arsenmal m shrimps Chemosphere, 11 (1982) 287. 4 J.R Cannon, J S Edmonds, K A. Francesconi, C.L. Raston, J.S. Saunders, B.W Skelton and A H White, Isolation, crystal structure and synthesis of arseno-betame, a constituent of the western rock lobster, the dusky shark, and some samples of h u m a n urine. Aust. J. Chem., 34 (1981) 787. 5 J B. Luten, G Riekwel-Booy and A. Rauchbaar, Occurrence of arsenic m plame
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(Pleuronectes platessa), nature of organo-arsenm compound present and its excretion by man. Environ. Health Perspect., 45 (1982) 165. 6 H. Yamauchi and Y. Yamamura, Metabolism and excretion of orally ingested trimethylarsenic in man Bull. Environ. Contam. Toxmol., 32 (1984) 682. 7 H Yamauchl and Y Yamamura, Metabolism and excretion of orally admmLstered dlmethylarslnlC acid m the hamster Toxmol. Appl. Phv.rmacol, 74 (1984) 134. 8 H Yamauchi and Y. Yamamura, Dynamic change of inorganm arsenm and methylarsenm compounds in human urine after oral intake as arsenic tmoxlde. Ind. Health, 17 (1979) 79 9 J P Buchet, R. Lauwerys and H Roels, Comparison of the urinary excretion of arsenic metabolites after a single oral dose of sodium arsenlte, monomethylarsonate, or dlmethylarsinate m man Int. Arch. Occup Environ Health, 48 (1981) 71 10 M. Vahter, Blotransformation of trivalent and pentavalent morgamc arsenm in mine and rats. Environ. R e s , 25 (1981) 286 11 F Bertolero, E. Marafante, J. Edel Rade, R Pietra and E. Sabblom, Blotransformatlon and lntracellular binding of arsenic m tissues of rabbits after intraperitoneal admmmtratlon of 74As labelled arsemte. Toxicology, 20 (1981) 35 12 E Marafante, F Bertolero, J Edel, R. Pietra and E. Sabbloni, Intracellular interaction and biotransformatlon of arsenite m rats and rabbits Sci Total Environ, 24 (1982) 27 13 H. Yamauchl, M. Iwata and Y. Yamamura, Matabolism and excretion of arsenic tmoxlde in rats. Jpn J Ind. Health, 22 (1980) 111 14 H. Yamauchl and Y. Yamamura, Urinary inorganic arsemc and methylarsemc excretion following arsenate-rich seaweed lngestmn Jp n J Ind Health, 21 (1979) 47
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