Toxicology, 63 (1990) 73--84

Elsevier Scientific Publishers Ireland Ltd.

Inorganic arsenic methylation by rat tissue slices

B. Georis, A. Cardenas*, J.P. Buchet and R. Lauwerys

Unit6 de Toxicologie lndustrielle et de M6decine du Travail, Catholic University o f Louvain, Clos
Chapelle-aux-Champs, 30.54. B-1200 Bruxelles (Belgium)
(Received December 5th, 1989; accepted March 23th, 1990)

Summary

Rat liver, kidney and lung slices methylate trivalent inorganic arsenic (Aslll) to monomethylar-
sonic acid (MMA) and dimethylarsinic acid (DMA); the liver has the greatest methylating capacity.
Aslll enters the liver cells by a diffusion process followed by extensive binding to intracellular
components which favors its extensive accumulation inside the cells. Reduced glutathione regulates
Aslll metabolism through several mechanisms: facilitation of Aslll diffusion into the cells, stimula-
tion of the first methylation reaction and increase of DMA excretion by the cells. An excess of Aslll
inhibits DMA production by liver cells but this inhibition is reversible; mercuric ions inhibit both
MMA and DMA production probably by decreasing inorganic arsenic (Asi) uptake and the second
methylation reaction. DMA can be produced from MMA by rat liver slices and this methylation step
is stimulated by GSH. In contrast to Aslli, AsV is not extensively taken up by the hepatocyte and is
thus poorly methylated.

Key words: Arsenic; Methylation; Rat tissues

Introduction

The biotransformation of inorganic arsenic (Asi) by mammals leads to the

production of monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)
which are mainly excreted in urine [1].
So far, the mechanism of Asi biotransformation has only been studied in vitro
with rat liver cytosol. It has been found that the addition of GSH and of S-
adenosyl-L-methionine (SAMe) as methyl group donor is required and that
vitamin B-12 enhances the methylating activity. In rat liver cytosol, an excess of
substrate and the addition of Hg 2" (0.1 mM) prevent the formation of DMA [21.
Furthermore, the methylation of Asi is slightly inhibited by the prior enrichment
of the incubation medium with MMA (feed-back inhibition) [3].
In the present study we have further investigated the mechanism of Asi methy-

Address all correspondence and reprint requests to: Prof. R. Lauwerys, Industrial Toxicology and
Occupational Health Unit, Clos Chapelle-aux-Champs, 30.54. B-1200 Bruxelles, Belgium *Research
fellow in Toxicology of the European Science Foundation.

0300-483X/90/$03.50
© 1990 Elsevier Scientific Publishers Ireland Ltd. 73
Printed and Published in Ireland
lation by using rat tissue slices which offer the advantage of preserving cell struc-
ture and may therefore be more representative of the in vivo situation than the
cytosolic preparation.

Materials and methods

Reagents
Sodium metaarsenite (AslIl) and sodium arsenate (AsV) were obtained from
Merck (Darmstadt, F.R.G.) and sodium methylarsinate (99.4070) from Carlo Erba
(Milano, Italy). Vitamin B-12, DL-buthionine (S,R) sulfoximine (BSO) and GSH
were purchased from Sigma Chemie Gmbh (Deisenhofen, F.R.G.). S-adenosyl-L-
methionine (SAMe), in the form of sulfuric and p-toluene suifonic acid salt, was
kindly offered by Bioresearch Co (Liscate, Italy). Tetra-n-butyl ammonium
hydroxyde solution (40070 in water) (TBA) and l-decanol were obtained from
Aldrich-Chemie (Steinheim, F.R.G.). Other chemicals were analytical grade
reagents from Merck (Darmstadt, F.R.G.).

Animals
Two- or 3-month-old male Sprague--Dawley rats (150--250 g) were used. The
animals were anaesthetized with pentobarbital (60 mg/kg, i.p.) and after section-
ing the inferior vena cava, they were perfused with ice-cold 0.907o NaCI through a
needle introduced in the left ventricle [3]. When lung preparations were used, the
renal artery was first cut and the lungs were rapidly perfused in situ through the
pulmonary artery [4].
In some experiments, the animals were injected i.p. with BSO dissolved in dis-
tilled isotonic water at a dose of 625 mg/kg, 3 h before sacrifice [51 in order to
decrease the GSH content of the liver. Control animals were given 2 ml NaCI
0.9°7o (0.15 M) i.p.

Slice preparation and incubation

The perfused organ (liver, lung or kidney) was directly transferred to a
Mcllwain tissue chopper (Mickle Laboratory Engineering, Co. Ltd, UK) to pre-
pare slices of 0.6 mm thickness [4].
About 70 mg of tissue (3 or 4 slices) were transferred to a plastic tube (30 x
70 mm) kept at 0°C and containing 3 ml of Krebs-Ringer phosphate buffer solu-
tion at pH 7.4 with glucose (11 mM), Asi and cofactors [4]. The tubes were
closed and placed in a shaking water bath. Incubation was carried out at 37°C
under N 2 or air. At the end of the incubation period, the slices were removed,
washed in buffer, blotted on filter paper and transferred in a tube containing 1
ml TBA (1607o) which was placed in a water bath at 80°C during 2 h. The incu-
bation medium was poured in a tube containing 50 /al concentrated HCI. The
amount of Asi, MMA and DMA was measured in both the slices and medium
preparation.

A nalyticai procedures
Inorganic arsenic (Asi) and its metabolites, MMA and DMA, were determined

74
by flameless absorption spectrometry as described previously [1] but with the fol-
lowing modifications: a decanol drop was added to the sample to avoid the for-
mation of foam during NaBH 4 addition and the U-shaped tube was warmed up
at ambient temperature to improve the separation of MMA and DMA peaks
from that of Asi.
For non-protein thiol determination, liver slices (100 mg) were placed in a tube
kept at 0°C to which were successively added 0.5 ml trichloroacetic acid (25°7o in
water) and 2 ml water. After thorough mixing, the tube was centrifuged at 1200 g
for 20 min. The thiol content of the supernatant was determined with Ellman
reagent [6]. The results are expressed as GSH equivalent since it is known that
the latter represents the major part of the low molecular weight thiols present in
the liver cytosol.
The leakage of LDH measured according to Korzeniewski and Callewaert [7]
from tissue slices to the incubation medium was used as an index of cell mem-
brane integrity [8]. The results are expressed in percent of total LDH activity
which was determined after ultrasonication of the slices during 3 min (Vinsonic
300, Virtis Company, power setting 3) and addition of Triton X-100 up to a final
concentration of 0.1%. Under the various experimental conditions selected for
the study, the percentage of total LDH activity released from the slices in the
incubation medium after 5 h incubation at 37°C is (mean __ S.E.) 15.4 _+ 1.1,
20.0 _+ 2.0 and 25.4 _ 3 for the kidney (n = 20), lung (n = 20) and liver (n =
16) respectively. We have also checked that LDH activity is not influenced by the
reagents added to the incubation medium.

Statistical analysis
The results were analysed by one- or two-way analysis of variance (differences
with P < 0.05 were considered statistically significant). In case of heterogeneous
variances, RANK and GLM procedures were used [9]. Comparisons between
means were performed with the D U N C A N ' S test and the paired t-test.

Results

The concentration of inorganic arsenic in the medium and in the liver slices
was compared after several incubation times with increasing concentrations of
AslII (Table I); this comparison was based upon the assumption that the density
of liver slices equals 1. The AsllI uptake by the slices is proportional to its
concentration in the incubation medium; the major part is taken up during the
first hour and at each concentration tested, the arsenic concentration is usually
more than 10 times greatei" in the slices than in the medium. Other experiments
have also shown that the inorganic trivalent arsenic uptake by liver cells does not
require oxygen, is temperature independent and that the cell membrane integrity
is not significantly changed even at the highest arsenic concentration tested (re-
sults not shown).
The comparison o f AsllI methylation by rat liver, kidney cortex and lung
slices is illustrated in Fig. 1. The addition of GSH to the incubation medium has
no effect on Asi methylation by lung slices but enhances the amount of MMA

75
 TABLE 1

INFLUENCE OF SUBSTRATE C O N C E N T R A T I O N AND INCUBATION TIME ON THE

UPTAKE OF AslII BY RAT LIVER SLICES

Incubation time (h) (Asi) tissue ~ M ) (Asi) medium (~M)

(Asi: l ~M)
1 4.1 ± 0.6" 0.3 ± 0.1
1.5 3.7 ± 0.4 0.2 ± 0.0
2 4.5 ± 0.4 0.2 ± 0.0
3 3.2 ± 0.4 0.2 ± 0.0
5 2.9 ± 0.3 0.2 ± 0.0

(Asi: 5/aM)
1 29.0 ± 2.0 1.5 ± 0.0
1.5 33.2 ± 1.5 1.7 ± 0.2
2 35.2 ± 1.6 1.4 ± 0.2
3 35.4 ± 2.0 1.1 ± 0.2
5 34.4 ± 1.8 1.1 ± 0.2

(Asi: 10 Iz M)
l 47.9 ± 1.5A b 5.3 ± 0.3
1.5 61.1 ± 4.0B 5.3 ± 0.1
2 67.1 ± 1.9 CB 4.7 ± 0.4
3 74.3 ± 2.8 C 4.2 ± 0.2
5 76.2 ± 0.5 C 3.6 ± 0.4

• Means ± S.E. of 5 experiments.

b Means with the same letter are not statistically different (Duncan test); no letter indicates no statisti-
cal difference.

Liver Kidney Lung

120

100 'B
O0
¢.D

<-'~
ID

80
MMA
B
•- ~
~
0
60 B
r--
-~ o 40 A A A
A

~ 20

0 J
contr GSH
SANe Hgz÷ conf.r GSH SANe contrGSH SAMe
+GSH +6SH +GSH
Fig. I. Influence of GSH, SAMe and Hg 2" on MMA and DMA production by rat liver, kidney cortex
and lung slices. The slices were incubated for 2 h in the presence of Asill (5 taM) with or without
GSH (5 mM), GSH + SAMe (1 mM) or Hg 2. (I mM) and the total amount (slice + medium) of
MMA and DMA was measured. Values are means ± S.E. of 5 experiments (For each tissue and
metabolite, means with the same letter are not statistically different).

76
TABLE 11

INFLUENCE OF GSH, SAMe AND Hgz" ON THE UPTAKE OF Aslll (5 ~M) BY RAT LIVER
SLICES

Incubation Asi taken up by tissue"

conditions (2 h) x 100
Asi in incubation medium

Control 36.4 ± 4.0* Bc

GSH (5 mM) 53.2 ___4.0 A
SAMe(I mM) + GSH(5mM) 25.4 ± 1.7B
Hg2" (I raM) 2.4 ± 0.1 C

• Asi taken up by tissue is the sum of Asi measured in tissue and of MMA and DMA measured in tis-
sue and in the incubation medium.
bMeans ± S.E. of 5 experiments.
Means with the same letter are not statistically different (P > 0.05, Duncan test).

and DMA produced by liver slices and decreases that produced by kidney slices.
In contrast to the results obtained with liver cytosol, the addition of SAMe to the
incubation medium is not required for the methylation of arsenic and even tends
to slightly decrease the methylation process by kidney and liver slices. Mercuric
ion inhibits both the production of M M A and DMA by liver slices while with rat
liver cytosol it had previously been shown to inhibit only DMA synthesis [2].
This discrepancy can be explained by the marked reduction of AsIII uptake by
liver cells in the presence of mercuric ions (Table It). Hence the decreased pro-
duction o f M M A by liver slices incubated in the presence of inorganic mercury is
due chiefly to a lack of substrate availability. The addition of GSH to the
medium slightly increases the total amount of AsIII which enters the hepatocytes.
Since it had been previously observed that high inorganic arsenic concentration
can decrease D M A production by liver cytosolic enzymes, we have attempted to
assess whether this phenomenon also occurs in intact cells. For this purpose two
series of liver slices were incubated with increasing concentrations of inorganic
arsenic, one series was kept in the same medium for the whole incubation period
while the other was transferred to an arsenic free medium 1 h after the start of
incubation. At various time intervals, the amount of MMA and DMA produced
was monitored (Figs. 2A,B). When arsenic is continuously present at a low con-
centration (l /~M) during the incubation period the amount of DMA produced
increases with time probably at the expense of the newly formed monomethylated
derivative, the concentration of which remains fairly constant during the whole
incubation period (Fig. 2A). At the same inorganic arsenic concentration (l /~M)
removal of the substrate after 1 h incubation has a rate limiting effect on MMA
and DMA accumulation during the next 4-h incubation period. At high arsenic
concentration, however, (10/~M) removal o f Asi from the medium after l h has
no influence on the total amount of methylated arsenic produced (MMA +
DMA) during the next 4 h but the proportion of DMA is much greater than

77
 O3 (/')
00[ i #M Asi 5 #M Asi
P1 10 #M Asi

200 F~DMA
(I.) '4--
0
,-~ C:D
>- E
c-
O
a~ o I00 I

CD~
c-

0
i 1.5 2 3 5 t t.5 2 3 5 t i.5 2 3 5

Time (hours)

300
b
I #M Asi 5 #M As i 10 #M As i
.i--q
< -~ 200
4~ D
MMA
loo
r--

t.5 2 3 5 i.5 2 3 5 1.5 2 3 5

Time (hours)
Fig. 2. Influence of substrate concentration and incubation time on MMA and DMA production by
rat liver slices, a: AsIII present in the incubation medium during the whole incubation period, b:
Aslll removed from the incubation medium after the first hour of the incubation period. At each
incubation time, the total amount (slice + medium) of M M A and D M A was measured. Values are
means ± S.E. of 5 experiments.

when As is continuously present in the medium. This indicates that in the pres-
ence o f 10 ~M Asi, the uptake o f Asi by the liver cells during the first hour o f
incubation is sufficient to allow the monomethyltransferase to work at maximum
velocity during the next 4 h but the continuous presence o f a high concentration
o f inorganic Asi inhibits the activity o f the methyltransferase which catalyzes the
production o f D M A (Fig. 2A).

78
T A B L E 111

EFFECT OF GSH D E P L E T I O N ON M M A A N D D M A P R O D U C T I O N FROM AslII BY RAT

LIVER SLICES

Incubation Total M M A ' ( M M A in tissue) Total D M A ' (DMA in tissue)

conditions (ng As/100 mg of tissue) (ng As/100 mg of tissue)

(Asi): 1 /aM
Control 13.4 ± 0.7 ( 12.2 - 0.3) 59.3 ± 9.6 (16.0 ± 1.6)
Control + GSH 25.9 _ 2.6 ( 13.3 ± 1.3) 70.7 ± 8.7 ( 3.2 ± 0.9)
BSO !1.0 ± 1.6( 9.4 ± 1.3) 47.5 ± 1.9( 5.9 _+ 0.3)
BSO + GSH 22.8 _+ 0 . 6 ( 13.3 ± 0.7) 63.8 ± 4 . 7 ( 3.6 ± 2.3)

(Asi): 5/aM
Control 129.3 ± 5.6 (110.6 ± 5.0) 169.4 ~ 14.0 (63.0 ± 4.4)
Control + GSH 143.5 • 16.6 ( 87.1 ± 9.6) 124.8 ± 24.9 ( 8.2 ± 2.9)
BSO 71.8 ± 3.7 ( 58.5 ± 3.5) 93.5 - 3.5 (23.3 ± 3.4)
BSO + GSH 135.8 ± 16.1 ( 71.9 ± 6.5) 120.4 ± 11.6 ( 8.0 ± 2.1)

(Asi): 10/aM
Control 142.7 + 27.3 (126.4 ± 24.0) 49.3 ± 16.4 (13.4 ± 4.0)
Control + GSH 283.5 ± 25.1 (158.1 ± 12.1) 76.9 + 15.6 ( 0.0 ± 0.0)
BSO 89.0 ± 3.0 ( 75.8 ± 3.5) 54.3 ± 4.9 (13.1 ± 4.4)
BSO + GSH 180.6 ± 9.2 ( i l 4 . 0 ± 6.5) 66.2 ± 4.2 ( 1.6 • 1.6)

Note: Rats (n = 4) were pretreated with BSO (625 m g / k g i.p.) and liver slices were incubated with
Aslll (1, 5, 10/aM) in the presence or in the absence o f GSH (5 raM) during 5 h (means ± S.E.).
•Sum of the a m o u n t present in the slices and in the medium.

The importance o f the role of GSH in the methylation of arsenic by rat liver
slices has also been assessed by pretreating the animals with buthionine sulfoxi-
mine (BSO, a gamma-glutamylcysteine synthetase inhibitor). The GSH level in
the liver of animals pretreated with BSO amounted to 30°70 of the amount meas-
ured in control animals (5.4 _+ 0.8/~mol/g liver, wet wt). MMA and DMA pro-
duction by liver slices was measured at three Asi concentrations in the absence
and in the presence of exogenous GSH (Table III). Generally, the pretreatment
with BSO results in a decrease o f arsenic methylation and the addition of GSH to
the medium tends to restore the production o f the methylated metabolites. The
results suggest, however, that it is the first methylation step which is mainly
affected by GSH depletion and that the reduced production o f DMA found at
the two lowest Asi concentration (47.5 vs. 59.3 ng As/100 mg o f tissue at 1 pm
Asi and 93.5 vs. 169.4 ng As/100 mg of tissue at 5 /zM Asi) is secondary to the
reduced production of the monomethylated metabolite since this phenomenon is
not observed (54.3 vs. 49.3 ngAs/100 mg of tissue) at the highest Asi concentra-
tion tested (10/~M). When animals have been pretreated with BSO the uptake of
Asi by liver slices (Table IV) is reduced and the uptake returns to normal or even
is stimulated when GSH is added to the incubation medium. It is interesting to
notice that both in control and in BSO pretreated livers, the addition of GSH to

79
 TABLE IV

EFFECT OF GSH DEPLETION ON THE Asill UPTAKE BY RAT LIVER SLICES

Incubation Asi taken up by tissue'

conditions x 100
Asi in incubation medium

(Asi): 1 gM
Control 51.0 ± 10.5b
Control + GSH 54.7 + 5.4
BSO 32.5 ± 3.4
BSO + GSH 52.2 _ 3.5

(Asi): 5 gM'
Control 40.3 ± 6.5
Control + GSH 58.9 ± 3.8
BSO 29.5 ± 3.5
BSO + GSH 47.7 + 4.8

(Asi): 10 ~M a
Control 28.2 ± 1.4
Control + GSH 41.9 _ 5.0
BSO 20.9 ± 2.1
BSO + GSH 35.0 ± 4.5

Note: The experimental conditions are similar to those described in the legend of Table III.
• Asi taken up by tissue is the sum of Asi measured in tissue, and of MMA and DMA measured in
tissue and in the incubation medium.
bMean _+ S.E. of 4 experiments.
' GSH effect, P -.- 0.003; BSO effect, P = 0.04; interaction, P = 0.92 by two-way anova.
"GSH effect, P = 0.003; BSO effect, P = 0.07; interaction, p = 0.91 by two-way anova.

MMA DMA
100
A

80
co o3

~,
t3~
60
r~
C:n
>- E

NS
~
:-- 20i:

mill
Aslll AsV Aslll AsV
Fig. 3. Influence of Asi oxidation state (5 gM) on MMA and DMA production by rat liver slices. The
slices were incubated for 2 h in the presence of Aslll or AsV (5 gM) and the total amount (slice +
medium) of MMA and DMA was measured. Values are means ± S.E. of 4 experiments (difference
between AsIIl and AsV: **P < 0.01; ns P > 0.05).

80
TABLE V

EFFECT OF GSH ON THE P R O D U C T I O N OF DMA FROM MMA

MMA DMA production" (ng As/100 mg of tissue)

concentration
(~M) Control + GSH

1 < 0.7 2.4 ± 0.6 TM

10 < 0.7 5.0 ± 0.7""
50 0.8 ± 0.6 14.2 ± 1.8""
100 4.4 ± 1.2 35.8 ± 4.3""

• Sum of the amount of DNA present in the slices and in the medium at the end of the incubation
period.
bDifference between GSH and control *P < 0.05; **P < 0.01, ***P < 0.001 (Student t-test).
Note: Rat liver slices were incubated with or without GSH (5 mM) during I h in the presence of
increasing concentrations of MMA (I, 10, 50, 100 taM) followed by 4 h incubation in a MMA free
medium (means • S.E. of 5 experiments).

the medium stimulates the transfer of DMA from the slices to the medium (Table
ill).
The valence state of arsenic has a major influence on the methylation of the
element by liver slices. When liver slices are incubated during 2 h with the same
concentration (5 /aM) of AsIIl or AsV a marked difference is observed with
regard to the production of M M A which is about l0 times higher with AsIII than
with the pentavalent form. The amount of DMA produced, however, is not sig-
nificantly different between both substrates (Fig. 3). It must, however be pointed
out that after an incubation period of 2 h, the amount of arsenic taken up by the
liver slices is about 8 times higher when the element is in its trivalent than in its
pentavalent state (results not shown). The need for pentavalent arsenic to be
reduced before its monomethylation and its limited uptake by liver cells are two
factors which explain the lower yield of M M A when the slices are incubated with
AsV.
Experiments were carried out to assess whether liver slices could use MMA for
the production of DMA. To be able to detect small amounts of DMA without
analytical interference of high concentrations of MMA, the slices were first incu-
bated during 1 h with increasing concentration of MMA (1, 10, 50 or 100/aM),
then transferred to a MMA free medium and incubated for a further 4-h period.
While the transformation of M M A into DMA can not be demonstrated with the
cytosolic enzyme system (results not shown), a clearcut production of DMA is
found when rat liver slices are incubated with MMA mainly when GSH is added
to the medium (Table V).

Discussion and conclusions

Trivalent Asi readily enters the liver cells (Table I). This uptake probably does
not require energy as it is independent of the presence of 02. A similar gradient-
dependent transfer of AsIII (diffusion) has also been observed with isolated

81
hepatocytes [9] and other mammalian cells such as Syrian hamster embryo cells
[10], mouse fibroblasts [11], mouse embryo cells [12], human fibroblasts and
Chinese hamster ovary cells [13]. The results obtained with rat liver slices incu-
bated in the presence of GSH and with slices obtained from animals pretreated
with BSO (Tables II and III), however, suggest that GSH may facilitate AslIl
uptake by hepatocytes (facilitated diffusion). Nevertheless, the inhibitory effect
of GSH depletion on inorganic arsenic methylation by rat liver slices cannot
simply be due to the reduced uptake of AsllI by the cells since we have pre-
viously shown with rat liver cytosol preparation that GSH is also required for the
first methylation reaction (MMA production) [31.
The present results and those of our previous studies [3,14] suggest that GSH
may influence AslII metabolism through several mechanisms facilitation of its
uptake by the cells, modulation of the first methylation reaction and stimulation
o f the excretion of DMA. By incubating tissue slices rather than cytosolic frac-
tions, it has been possible to unequivocally demonstrate that tissues other than
the liver, e.g. lung and kidney cortex, can methylate inorganic trivalent arsenic.
On the basis of the relative organ weight and substrate (Aslll) availability in
vivo, the total amount of methylated arsenic which can be produced by the liver
amounts roughly to 20 times that produced by the lungs which is in turn three
times higher than that synthesized by the kidneys. In contrast with the results
obtained with liver cytosolic preparations, Asi methylation by liver slices does not
require the addition of SAMe which can be synthesized by the cells as long as
ATP and methionine are available [14al.
In the cytosolic system, mercuric ion inhibits the second methylating step only
[2] while it reduces the production of both methylated arsenicals by liver slices
(Fig. 1). In the latter case, however, the decreased MMA synthesis can be
explained by the marked reduction of Asi uptake by the cells when the slices are
incubated in the presence o f Hg 2" ions (Table II). Mercuric ion can thus decrease
the rate of MMA production by inhibiting Asi uptake by the cells and, when pre-
sent in the ceils, by directly inhibiting the second methylation reaction.
The production of DMA by liver slices is also inhibited by an excess of AslII
which is in agreement with our previous observation with rat liver cytosol [2].
However, this inhibitory effect of AsllI on the second methylation step is
reversible since DMA production returns to a normal rate when AsllI is removed
from the medium (Fig. 2b).
The central role of GSH in arsenic metabolism may explain our previous find-
ings in patients with liver insufficiency administered a small intravenous bolus of
AslII since it is known that hepatic GSH may be reduced in such patients
[15,16]. The reduced AslIl uptake has decreased the amount of monomethylated
metabolite produced but the latter has been transformed into DMA to a greater
extent than in control subjects because the second methylation reaction was less
inhibited by AslII; hence our observations of a decreased urinary excretion of
MMA and an increased excretion of DMA following a single administration of
AslIl to patients with liver diseases [17]. It is also worth mentioning that
glutathione level and glutathione-S-transferase activity are increased in arsenic-
resistant Chinese hamster ovary cells [18] and that in asynchronous human fibro-

82
blasts, G1 phase and asynchronous Chinese hamster ovary cells the intracellular
level o f GSH is negatively correlated with the cytotoxic effect of sodium arsenite
[13]. The resistance to AslII toxicity associated with an increased intracellular
GSH level may partly result from its stimulating effect on AslII
monomethylation. When the slices are incubated with AsV the synthesis of meth-
ylated derivatives is much lower than with trivalent Asi. This is due to the lower
substrate availability rather than to the rate of reduction of AsV. Indeed,
although AsV has to be reduced to AslII before being methylated [191, it appears
that mammalian cells exposed to AsV quite easily converts most of the cytosolic
arsenic to the trivalent form [12]. According to these authors, this reduction
partly depends on the availability of reduced glutathione. Moreover results of
Table III suggest a similar possible role for GSH in the synthesis of DMA from
MMA. According to the reaction mechanism proposed by Cullen et al. [20] a
reduction step of MMA is also needed before the introduction of a second methyl
group to form DMA (nucleophilic attack by a trivalent arsenical on the C - - S
bond of SAMe); the electrons needed for this reduction could also originate from
the cellular GSH pool. The finding of a production o f DMA from MMA by rat
liver slices, a reaction which could not be identified by using a rat liver cytosolic
preparation [2] is in agreement with our previous observations in volunteers who
were found to excrete DMA in urine following oral administration of MMA [21].
In conclusion, the pathway that we have recently proposed for the inorganic
arsenic biotransformation by mammalian liver [3] is confirmed by the present
study with rat liver slices. It, however, allows to add four new informations, i.e.
GSH facilitates the uptake of AslII by the hepatocytes, MMA can be trans-
formed into DMA, GSH is not required for the second methylation reaction
when AslII is used as substrate, but stimulates DMA excretion from the cells.
Further study is required to confirm whether the increased AsllI methylation
found in some mammalian cells made resistant to AslII [11] only results from an
increased GSH production and glutathione-S-transferase activity or whether some
methyltransferases are also involved.

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