The effect of methyltransferase inhibition on the metabolism of [74As]arsenite in mice and rabbits

Chem -B~ol.Interactions, 50 (1984) 49--57 Elsevier ScientificPublishers Ireland Ltd

THE E F F E C T OF METHYLTRANSFERASE INHIBITION ON METABOLISM OF [ ~As] ARSENITE IN MICE AND RABBITS*

ERMINIO MARAFANTE

TM

49

THE

and M A R I E V A H T E R b

aRad~ochem~stry Division, C E C Join.t Research Centre, 1-210 20 Ispra (Italy)and bNational Institute of Enwronmental Medicine, P 0 Box 602 08, S-I04 01 Stockholm (Sweden)

(Received December 9th, 1983) (Revision received February 9th, 1984) (Accepted February 20th, 1984)

SUMMARY The effect of perlodate-oxldized adenosine (PAD), an inhibitor of certain methyltransferases, on the biotransformation and tissue retention of [V4As]arsenite in mice and rabb~ts was studied. Injection of PAD (100 /~mol/kg body wt.), 15 rain prior to the injection of [~4As]arsenite (0.4 mg As/kg body wt.), resulted in a 25--70% decrease in the production o f [74As]dimethylars~nic acid ([7~As]DMA). This implies that S-adenosylmethlonine ~s the methyl-donor in the methylat~on of inorganic arsenic ~n vivo. Due to lnterachon of the u n m e t h y l a t e d arsemte with tissue constituents the PADtreated animals had significantly higher {2--6 times) tissue concentrations of V4As than d~d the controls. This effect was first observed in the liver, indicating that th~s organ m the main site of the methylat~on of arsenic. The increase ~n the tmsue retention due to the PAD-treatment remained also after cessation of the ~nhibition of methylation. The results can be seen as confirmation that alkylation of inorganic arsenic acts as a detomfication mechanism in mammals.

Key" words" Methyltransferase inhibition -- Arsenite -- Methylatlon -Detoxlficatlon

*This study was performed within the framework of collaboration No. 1644-81-10TS ISP S between the Commission of the European Communities and the National Instituteof Environmental Medicine, Stockholm, Sweden. **Present address National Instituteof Environmental l~ediclne,P.O Box 602 08, S-104 01 Stockholm, Sweden. Abbreviahons D M A , dimethylarsinicacid,PAD, periodate-oxidizedadenosine.

0009-2797/84/$03.00 © 1984 Elsevier Sclentifm Pubhshers Ireland Ltd. Printed and Published ~n Ireland

50 INTRODUCTION In man and most experimental ammals DMA ~s the major arsemc metabol~te in urine following exposure to inorgamc arsemc [1--8]. The m e t h y l a t m n is considered to be a detoxification process, s~nce the methylated arsemc has a low affimty for t~ssue const~tutents and ~s readily excreted as such m the urine [9--11]. Accordingly, the marmoset m o n k e y which ~s unable to methylate ~norgamc arsemc, shows considerably longer r e t e n t m n of arsemc than do other species [12]. The site and mechamsm of the m e t h y l a t m n ~n mammals are not y e t known. Although ~ntest~nal bacteria from rats are able to methylate inorgamc arsemc when incubated ~n wtro [13], ~t has been shown t h a t the intestine is not the main s~te of the methylat~on ~n v~vo [14]. S~nce the arsemc administered orally is methylated to a h~gher degree than that entenng the body parenterally [2,7], ~t seems hkely t h a t the hver ~s one ~mportant s~te of the methylat~on. In fact, methylated forms of arsenic have been detected following in vitro lncubat~on of inorgamc arsemc w~th rat hver cells [15]. It has been suggested that the enxqronmental methylat~on of metalloids occurs by nucleophilic or free radical attack by metalloid salts of lower oxldat~on state on S-adenosylmethiomne or methyl-B~2 [16]. The mm of the present study was to ~nvestigate the influence of PAD, an ~nh~b~tor of certain methyltransferases [17], on the metabolism of [V~As]arsemte ~n m~ce and rabbits. MATERIALS AND METHODS

[74As]arsenite was prepared by adding [74As]arsemc acid (Amersham International, UK) to a water solution of sodium arsemte, which was then reduced and controlled as described by Vahter and Norln [18]. PAD was prepared according to Hoffman [17]. A solution (0.5 M) of adenosine m 0.1 M acetic acid (pH 4.0) was oxidized with 0.5 M NaIO4, adjusted to pH 7 w~th NaOH and kept overmght in a refrigerator. The precipitated PAD was separated by centr~fugat~on, washed three t~mes with water, and dried in a desmcator. The concentration of PAD ~n the solutmns was determined by measunng the absorbance at 259 nm (pH 7), using a molar extinction coefficient of 15 400 M -~ cm -~. Solutions (0.1 mmol/ml) ~n NaC1 {0.9%, w/v) were prepared ~mmedlately before use. Groups of 8 male m~ce (NMRI, about 30 g) and 3 male rabb~ts {New Zealand White, 2.5 kg) were g~ven e~ther ~ntrapentoneal lnject~ons of 100 pmol PAD/kg body wt., 15 m~n before an intravenous mject~on 0n the taft vein of the m~ce and the ear vem of the rabbits) of [7~As]arsemte {0 4 mg As/kg body wt.), or the [7~As]arsenite only {controls). The animals were kept for 16--72 h ~n metabohc cages designed for separatmn of feces and unne. Food and d n n k m g water were g~ven ad lib~tum. Other groups of 8 m~ce and 3 rabb~ts, given the same doses of PAD and arsemte or arsemte only, were sacrificed at d~fferent t~mes after the ~As ~nject~ons (1 and 16 h for the m~ce

51 and 16 and 72 h for the rabb~ts). Blood, liver, kidneys, lungs, epididym~s and a port~on of the skin were removed and measured for ~4As content. Subcellular fractions of the hver were obtained by differential centrifugation after homogemzat~on ~n 0.01 M Tris--C1 buffer (pH 8.1) containing 0.25 M sucrose. The crude nuclear (10 min at 700 × g), mitochondrial (10 rain at 9000 × g), lysosomal and microsomal (90 rain at 105 000 × g) fractions obtmned were further purified by 2-fold washing in Tris--C1 buffer followed by centnfugat~on. Gel chromatographic separation of the mouse liver and k~dney cytosols were carried out on columns ~2.5 × 100 cm) of Sephacryl S-200 (Pharmac~a, Uppsala, Sweden). Separatmn of the ~"As metabolites ~n urine was performed on ion exchange columns of AG 50W X4, 100--200 mesh (Bio-Rad, U.S.A.) according to the method of Tam et al. [19], by which inorganic arsenm, methyharsomc acid and DMA are sequentially eluted with 0.5 M HC1, H:O and 4 M NH4OH. The ~"As-act~vity in the samples was measured using gamma scintillation counting (Packard A5320 Autogamma). The amount of arsenic present ~n the samples was determined by comparision w~th standard solutions of known specific act~wty. RESULTS

The unnary excretion of [74As]arsemc was considerably lower in mice and rabbits treated with PAD prior to the administration of [~4As] arsenite than in m~ce and rabbits g~ven [7"As]arsemte only (Fig. 1). This was due to the lower excretion of [V"As]DMA in the PAD-animals compared to the controls {P < 0.01 for the rabb~ts), while the excretion of inorgamc arsenic was essentially the same ~ndependent of the treatment. The effect of the PAD on the unnary excretion of DMA was most pronounced dunng the first day after admimstrat~on (Fig. 2). The fecal excretion of [V"As]arsemc was less than 4% of the dose, ~ndependent of treatment and ammal species. The effect of the pretreatment with PAD on the tissue concentrations of 7"As ~s shown ~n Figs. 3 and 4. One hour after the admimstration of the [ ~4As] arsemte the PAD-treated mice showed h~gher concentrations (P < 0 . 0 5 ) of ~"As ~n the hver, about the same in kidneys and skin and lower (P < 0.01) in the blood and the lungs than the control mice (Fig. 3). At 16 h the PAD-treated mice had 2--3 t~mes higher (P < 0.01, except the hver) 7"As-levels than the controls in the tissues as well as in the blood. In the rabb~ts {Fig. 4) the d~fferences in tissue retention of V"As in relation to the PAD-treatment were shown to remain for at least 3 days. At that t~me the PAD-rabb~ts had 4--6 times higher tissue levels o f V"As than the control rabbits (P < 0.05 except the lungs). The subcellular d~stnbutlon of V4As in the hver of the m~ce was not effected by the PAD-treatment (Table I). About 50% of the V"As was found in the soluble cytoplasmic fraction and 20--30% in the nuclear fraction, independent of the treatment.

52 urinary

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F~g 1. Effect of P A D on the urlnary excretion of 74As as inorgamc arsenic and D M A in mice and rabbits after Lv. adm~mstratlon of [7'As]arsenite. M e a n + S.E. of 3 rabbits and 2 groups of 4 rmce each. P A D , given P A D 15 rnin before injection of [~'As]-arsemte, C, controls given [~4As]arsenite only.

F~g. 2. Effect of PAD on the daffy urinary excretion of [7'As]DMA in rabbits following i v a d m i m s t r a t i o n o f [ 74As]arsenite. Mean ± S E. o f 3 rabbits. • • , given P A D 15 mm before injectmn of [ ~4As]arsemte, •------•, controls given [ ~As]arsemte only.

The gel chromatography of the liver and kidney cytosols of the PADtreated mice (Ftg. 5) revealed a distribution of ~4As among high molecular weight c o m p o n e n t s (Ve/Vo 1--2) similar to that of the control mine. In the low molecular weight region (Ve/Vo 3--4}, however, the PAD-treated mice showed a higher ratio of peak B/peak A than the controls (Fig. 5). This was most hkely due to the decrease in the DMA production in the PAD mice, since the cahbration of the columns for the elution volume of [74As]arsemte, [ ~ A s ] a r s e n a t e and [7~As]DMA showed that DMA and arsenate were eluted at a Ve/Vo of a b o u t 3.1 (peak A), while the arsenite was eluted at a Ve/Vo of a b o u t 3.8 (peak B). DISCUSSION Transmethylatlon reactions involving S-adenosylmethlonine result in the formation of S-adenosylhomocysteine. Injection of PAD to mice causes a pronounced increase (about 50-fold) in liver S-adenosylhomocysteine, probably by d~rect inhibition of S-adenosylhomocysteine hydrolase [ 17,20].

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F i g 4 E f f e c t o f P A D o n t h e t i s s u e c o n c e n t r a t i o n o f ~4As ~n r a b b i t s , 1 6 a n d 7 2 h after 1.v a d m i m s t r a t m n o f [ ~ A s ] a r s e n i t e M e a n + S E o f 3 rabbits.

Such an accumulation of S-adenosylhomocyste~ne strongly ~nhlbits the transmethylation from S-adenosylmethiomne [ 21 ]. The present study shows that PAD also impairs the methylat~on of inorganic arsenic m mice and rabbits g~ving evidence for S-adenosylmethionine being the methyl-donor in the ~n vivo production of DMA, the mmn metabohte of inorgamc arsenic. The decrease ~n the DMA-productlon lasted for about one day after the adm~mstration, which fits well with the duration of the inhibitory effect of PAD on the S-adenosylhomocysteine hydrolase [ 17 ]. TABLE I EFFECT OF PAD PRETREATMENT ON THE SUBCELLULAR DISTRIBUTION OF ~4As (% O F ~4As IN T O T A L H O M O G E N A T E ) IN L I V E R O F MICE, I A N D 16 H A F T E R A D M I N I S T R A T I O N O F [~4As] A R S E N I T E Mean + S E. of 4 samples (tissues of 2 mice pooled) PAD, pretreated m~ce, C, control mice Fractmn

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Alkylation and dealkylation reactions are of fundamental importance in the metabolism of various toxic metals [22]. The results o f the present study can be taken as confirmation of the hypothesis that the methylation of inorgamc arsenic is a detoxification process, since the impairment of the methylation increased the tissue retention of arsenic. Most likely, this was caused by interaction of u n m e t h y l a t e d arsenite with tissue constituents, because arsenite has been shown to have a much higher affinity for cellular components than DMA [10]. Further support for this premise is the low a m o u n t of [ V4As] DMA in relation to the [ ~4As] arsenite in the liver cytosol of the PAD mice at 1 h. The increase in the tissue retention of 74As was shown to remain m the rabbits even after cessation of the effect of PAD on the methylation. The effect of PAD on the tissue retention of ~As was first observed in the hver, gw~ng support to the assumption that this organ is the main site of the methylation of arsemc. The inhibition of the methyltransferase activity in the hver by PAD probably decreased the a m o u n t of arsenic which was methylated and thereafter cleared to the blood. This was reflected in the low blood levels of ~4As in the PAD mice at I h after administration. At 16 h, when the effect of the PAD had almost ceased, the restarted methylation gave rise to higher blood ~"As levels in the PAD mice than in the control mice. The relatively high concentration on 74As in the lungs of the control mice compared to the PAD mice at I h was probably related to the preferenhal locahzation of DMA in the lungs [11,23].

56 The present findings will be of tmportance for the evaluation of factors ~nfluenc~ng the metabolism and toxicity of ~norgamc arsemc. For example, it has been shown t h a t diets low in meth~onine or chohne, leading to m e t h y l ~nsuffic~ency ~n rats, caused decreased S-adenosylmethiomne and ~ncreased S-adenosylhomocyste~ne concentrations in the hver [24]. S~nce the present study indicates that the liver ~s the mmn s~te of m e t h y l a t l o n of arsenic, ~t can be anticipated t h a t such diets would also decrease the methylation of ~norgamc arsemc, thereby lncreas~ng ~ts toxicity. It has earher been shown that differences in the degree of ~ntracellular binding of arsenic between anunal species may be related to d~fferences ~n the rate of methylation [10]. It ~s possible that the d~fferences ~n the efficiency of the m e t h y l a t m n of inorgamc arsenic between ammal species ~ncluding man are, at least partly, due to differences ~n the transmethylase activity. There may, however, be other rate-lmalting steps ~n the detox~ficat~on reactions, s~nce arsenic is a substrate for oxidation-reduction reactions ~n addition to the alkylat~on reactions [25,26]. REFERENCES I T.J. Smith, E.A. Crecehus and J.C. Reading, Airborne arsenic exposure and excretion of methylated arsenic compounds, Environ Health Perspect., 19 (1977) 89. 2 S M. Charbonneau, G K.H. Tam, F. Bryce, Z Zawidska and E. Sandl, Metabolism of orally administered Inorgamc arsenic in the dog, Toxicol. Lett., 3 (1979) 107. 3 S M. Charbonneau, J.G Holllns, G.K.H. Tam, F Bryce, J.M R1dgeway and R.F. Willes, Whole-body retention, excretion and metabohsm of [74As|arsenlc acld ~n the hamster, Toxicol. Lett., 5 (1980) 175. 4 G.K.H Tam, S.M. Charbonneau, F. Bryce, C. Pomroy and E Sandi, Metabolism of ~norgamc arsenic (74As) in humans following oral ingestion, Toxicol Appl. Pharmacol , 50 (1979) 319. 5 J.P Buchet, R. Lauwerys and H. Roels, C o m p a r m o n of several methods for the determination of arsenic compounds in water and In urine, Int. Arch Occup. Envlron. Health, 46 (1980) 11. 6 F. Bertolero, E. Marafante, J. Edel Rade, R. Pletra and E Sabblom, Biotransformation and intracellular binding of arsemc ~n tissues of rabb~ts after intraperltoneal admlnistrat~on of 7~As labelled arsemte, Toxicology, 20 (1981) 35 7 M. Vahter, Biotransformatmn of tr~valent and pentavalent inorganic arsenic In m~ce and rats, Environ. Res, 25 (1981) 286. 8 E. Marafante, F. Bertolero, J. Edel, R. Pietra and E. Sabblom, IntraceIlular interaction and biotransformation of arsemte in rats and rabbits, ScL Total Environ, 24 (1982) 27 9 J.P. Buchet, R. Lauwerys and H. Roels, Comparison of the urinary excretion of arsenic metabohtes after a single oral dose of sodium arsemte, monomethylarsonate, or dlmethylarsinate in man, Int. Arch. Occup. Environ Health, 48 (1981) 71 10 M. Vahter and E. Marafante, Intracellular interaction and metabolic fate of arsemte and arsenate ~n mice and rabb~ts, Chem. Biol. Interact., 47 (1983) 29. 11 M. Vahter, E Marafante and L. Dencker, Tissue distribution and retention of ~4Asd~methylarslnic acid in mice and rabbits, Arch. Envlron. Contain Toxicol , 13 (1984) in press. 12 M. Vahter, E. Marafante, A. Lindren and L. Dencker, Tissue distribution and subcellular binding of arsenic in marmoset monkeys after injection of ~4As-arsenite, Arch Toxicol , 51 (1982) 65.

57 13 I.R. Rowland and M.J. Davis, In wtro metabolism of inorganic arsenic by the gastrointestinal microflora of the rat, J. Appl. Tox~col., I (1981) 278. 14 M. Vahter and B. Gustafsson, Biotransformation of inorganic arsenic in germfree and conventional mice, in M. Anke, H.-J. Schneider and C. Bri~ckner (Eds.), Proceedings of 3rd Symposium on Trace Elements, Arsenic, Abteilung Wissenschaftliche Pubhkationen der Friedrich-Schiller-Universifiit,Jena, DDR, 1980 p. 123. 15 S.A. Lerman, T.W. Clarkson and R.J. Gerson, Arsenic uptake and metabolism of liver cells is dependent on arsenic oxidation state, Chem.-Biol. Interact., 45 (1983) 401 16 J M Wood, A Cheh, L.J. Dizikes, W.P. R~dley, S. Rakow and J.R Lakowicz, Mechanism for the biomethylation of metals and metalloids, Fed. Proc., 37 (1978) 16. 17 J.L. Hoffman, The rate of transmethylation ~n mouse liver as measured by trapping S-adenosylhomocysteine, Arch. B~ochem Biophys., 205 (1980) 132. 18 M. Vahter and H. Norin, Metabolism of ~4As-labeled tr~valent and pentavalent inorgamc arsemc in mice, Enwron. Res., 21 (1980) 446. 19 G.K H. Tam, S M. Charbonneau, F. Bryce and G. Lacroix, Separation of arsenic metabol~tes m dog plasma and urine following intravenous injection of ~4As, Anal. Biochem , 86 (1978) 505. 20 J.L. Hoffman, ~n E. Usdin, R.T. Borchardt and C.R. Creveling (Eds.), Transmethylation, Elsevier, Ameterdam, 1979, p. 99. 21 W.K Pa~k and S. Kim, Protein methylatlon, in" A. Meister (Ed.), Biochemtstry, A Ser~es of Monographs, John Wiley and Sons, New York, 1980. 22 T.W. Clarkson, Effects - - general principles underlying the toxic action of metals, in L. Fr~berg, G.F. Nordberg and V.B. Vouk (Eds.), Handbook on the Toxicology of Metals, Elsevier, Amsterdam, 1979, p. 99. 23 A. L~ndgren, M. Vahter and L. Dencker, Autorad~ographic studies on the distribution of arsemc ~n mice and hamsters administered ~4As-arsenite or -arsenate, Acta Pharmacol Toxlcol., 51 (1982) 253. 24 N. Shivapurkar and L.A. Poir~er, Tissue levels of S-adenosylmethionine and S-adenosylhomocysteine ~n rats fed methyl-deficient, amino acid-defined diets for one to five weeks, Carcinogenesis, 4 (1983) 1051. 25 B C. McBride and R.S. Wolfe, Biosynthesis of dimethylars~ne by methanobactermm, Biochemistry, 10 (1971) 4312. 26 M Vahter and J. Envall, In vivo reduction of arsenate in mice and rabbits, Environ. Res., 32 (1983) 14