Pharmacology & Toxicology 1992, 70; 352-356.

Effect of Various Antidotes on Biliary Excretion of Arsenic in

Isolated Perfused Livers of Guinea Pigs after Acute
Experimental Poisoning with As,O,*
F. X. Reichl, H. Miickter, H. Kreppel and W. Forth
Walther Straub-Institut of Pharmacology and Toxicology, Ludwig-Maximilians-Universityof Munchen,
NuDbaumstrasse 26, D-8000 Miinchen, Germany
(Received August 19, 1991; Accepted November 14, 1991)

Abstract: The effect of the dithiols British Anti-Lewisite (BAL), dimercaptopropanesulfonicacid (DMPS), dimercaptosuc-
cinic acid (DMSA) and a new metal binding agent 2,3-bis-(acetylthio)-propanesulfonamide (BAPSA) on the biliary
excretion of arsenic in perfused livers of guinea pigs after acute experimental poisoning with Asz03 was investigated.
Guinea pigs received As201, 10.0 mg/kg subcutaneously at 9 a.m. as a single injection. One hour after the injection the
livers were perfused (2.5 ml x min.-' x g-' liver) with Krebs-Henseleit buffer and glucose for 80 min. After 40 min. of
saline perfusion (control) 0.1 or 0.7 mmol/l BAL, DMSA, DMPS, or BAPSA were added to the perfusate and arsenic
elimination in the bile and effluent perfusate was measured. The biliary excretion of arsenic in control livers between 40
and 80 min. was 0.7% of the total arsenic liver content before perfusion (=arsenic liver content after perfusionfportion
excreted in the bile+perfusate). After antidote addition (0.1 mmol/l) the excretion was 0.2% for livers perfused with
BAL, 6.8% for DMSA, 10.6% for DMPS, and 11.l% for BAPSA, respectively. After 0.7 mmol/l antidote the excretion
of arsenic was 0.1% in livers perfused with BAL, 9.6% for DMSA, 12.3% for DMPS, and 13.3% for BAPSA, respectively.
Except BAL, all compounds and most effectively BAPSA increased biliary excretion of arsenic. This indicates that
excretion of arsenic which normally is mainly renal is shifted towards faecal excretion by the dithiols.

Arsenic is mainly eliminated by renal excretion (Ducoff et al.

Materials and Methods
1948; Yamauchi & Yamamura 1985). Maehashi & Murata
(1986) proposed that although arsenic is mainly excreted by Animds. Male guinea Pigs (Wilab; Riedl, Miinchen, Germany),
the kidney, treatment with chelating agents may cause a 350-450 g fed standard diet (Alma, H 2011, Botzenhardt KG,
Kempten, Germany) and water ad libitum were used. The animals
substantial shift to faecal elimination of the toxicant by an were allowed to acclimatizefor at least Seven days.
increase in biliary excretion.
The liver is the target organ in acute poisoning with As,O, Chemicals. Arsenic trioxide (AszOl, Merck, Darmstadt, Germany);
where arsenic affects the carbohydrate metabolism (Reichl Urethane, 25% W ~ inV saline (Merck, Darmstadt, Germany); DL-
2,3-dimercaptopropanol (BAL, molecular weight 124.1) (Serva, Hei-
et al. 1988). Arsenic inhibits the activity of intramitochond- delberg, DL-2,3-dimercaptopropanesulfonate sodium
enzymes as pyruvate-dehydrogenase (PDH) (Peters salt x H,O (DMPS, molecular weight 228.3) (Hey1 & Co., Berlin,
1955) and a-ketoglutarate-dehydrogenase (a-KG-DH) Germany); meso-2,3-dimercaptosuccinicacid (DMSA, molecular
(Webb 1966) in the liver. weight 182.2) (EGA-Chemie, Steinheim/Albuch, Germany); 2,3-
The cont&,ution of biliary excretion to the reduction of bis(acetylthio)-propanesulfonamide (BAPSA, molecular weight
271.4) (Muckter, unpublished results); peanut oil (oleum arachidis,
the body Or liver arsenic burden was shown to become p.a.); ethanol bur, Merck, Darmstadt, Germany). All chemicals
substantially increased after treatment with some dithiols were analytical grade products.
(Kreppel et al. 1990). The difference in the pattern of eff-
cacy was proposed to be caused by different lipid solubility
and different distribution of the dithiols in organs. Table 1.
In the present paper the effect of the chelating agents on Treatment of nine randomized groups of each six animals.
the biliary excretion of arsenic was investigated by perfusion Concentration
of livers of guinea pigs with British Anti-Lewisite (BAL), of antidote Number of
dimercaptopropanesulfonicacid (DMPS), dimercaptosucci- Group Treatment Antidote (mmol/l) animals
nic acid (DMSA), and a new metal binding agent 2,3-bis(ac- Group 1 - - 6
ety1thio)-propanesulfonamide (BAPSA) after acute experi- Group 2 BAL 0.1 6
mental poisoning with As203. Group 3 DMSA 0.1 6
Group 4 DMPS 0.1 6
Group 5 BAPSA 0.1 6
Group 6 BAL 0.7 6
Group 7 DMSA 0.7 6
* Dedicated to Professor Dr. W. Rummel, HomburgISaar, on the Group 8 DMPS 0.7 6
occasion of his 70th birthday, October 23, 1991. GrouD 9 BAPSA 0.7 6
 ANTIDOTES ON BILIARY ARSENIC EXCRETION 353
Experimental protocol. After randomization in nine groups of six % arsenicelimination
animals each, guinea pigs were treated as shown in table 1. 18-, inthebile
All guinea pigs received As,O, subcutaneously as a single injec-
tion, 10 mg/kg, at 9 a.m. One hr after the injection the livers were
removed in anaesthesia (1 mg/kg urethane 25%, injection volume
4 ml/kg urethane) and perfused in a non-recirculating system
(Scholz ef al. 1973). The perfusion technique was described else-
where (Scholz 1968; Scholz et al. 1969). Krebs-Henseleit buffer
(Krebs & Henseleit 1932) containing glucose as substrate (10 mmoli
I), according to Kuhn & Scholz (1982) was used for the perfusion.
The perfusate was saturated with 95% 0 2 / 5 % C 0 2 at 37" and
pumped (2.5 ml x min.-' x g-' liver) for 80 min. through the liver
via a cannula placed in the portal vein.
The effluent perfusate was collected from the cannulated vena
cava and passed an oxygen electrode (teflon-shielded platinum elec-
trode, wire diameter 5 pm; Rank Brothers, Bottisham, England).
After ligation of ductus cysticus a cannula was placed in the 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0
ductus choledochus. Samples of the effluent perfusate and bile were time (min)
taken every 10 min. A 40 min. control perfusion period was run in Fig. 2. Arsenic elimination in the bile during 40 min. of perfusion
order to assess the spontaneous release of arsenic and remove the before and after addition of antidotes (0.7 mmol/l), in percent of
easily available toxicant as well as to stabilize the physiological
the total arsenic liver burden before perfusion. Experimental details
parameters (lactate and pyruvate eMux and oxygen consumption) see table 2.
and remove the anaesthetic (Thurman & Scholz 1973). After this
period either DMPS or DMSA (both dissolved in Krebs-Henseleit
buffer), BAL (dissolved in peanut oil and diluted with Krebs-Hense-
kit buffer, 2.9%), or BAPSA (dissolved in ethanol and diluted with
Krebs-Henseleit buffer, 0.14 or 1 mmol/l ethanol), were added to at 164" for 2.4 hr. The arsenic contents of the effluent perfusates
the perfusate for 40 min. were measured without HNO, treatment.
The efficacy of antidotes was calculated as difference between Arsenic content was measured as ASH, after treatment of the
the cumulative arsenic elimination 40 min. before and 40 min. after samples with NaBH, (3% w/v in 1% w/v NaOH, Riedel de Haen,
antidote addition compared to the difference in controls. Seelze, Germany) in an atomic absorption spectrometer (MHS 20,
The effects of two concentrations of each antidote were tested, Perkin-Elmer, Uberlingen, Germany).
The total arsenic content in livers before perfusion was calculated
0.1 and 0.7 mmol/l, respectively. The solutions of the thiol com-
as the sum of the arsenic portions excreted via bile+effluent perfu-
pounds were prepared daily immediately before use.
Livers of control animals were perfused with Krebs-Henseleit +
sate tissue liver content after eighty minutes of perfusion.
buffer only. One hr after acute experimental poisoning with As,O, Lactate and pyruvate were determined enzymatically according
high amount of arsenic was found in the liver (Reichl et al. 1988). to Bergmeyer (1986).
To be sure that the toxicant had reached the targets during
treatment with antidotes livers were therefore perfused I hr after
Statistical evaluation. Statistical significance of the differences be-
the injection with As,O,. tween the experimental groups was checked by the Wilcoxon-Mann-
Whitney test (a=O.OS, two tailed) (Sachs 1978).
Determination of arsenic. For the determination of arsenic in bile
and liver tissue, 0.3 ml (bile) or 0.3 g (liver), respectively, were Results
heated with 1 ml HNO, (65% w/v, Merck, Darmstadt, Germany)
A dose dependent increase in biliary arsenic excretion was

-
observed with DMSA, DMPS and BAPSA (fig. 1 and 2).
% atsenicelirnination The total arsenic content of the livers ranged from 450 to
In the bile 650 pg Adliver (meanfS.E.M. 508.8+ 10.0 pg Adliver)
the variation being similar in all groups.
Livers were assured to be in the metabolic steady state
I after 40 min. of perfusion where the lactate and pyruvate
efflux was 144+12 or 15+2 nmolxmin.-'xg-' liver, re-
spectively, and the oxygen consumption was 1020 95 *
nmol x min.-' x g-' liver (mean fS.E.M.). No effect on lac-
tate-, pyruvate-efflux or oxygen consumption was observed
in perfusion experiments with Krebs-Henseleit buffer after
addition of peanut oil (2.9%) or ethanol (0.14 or 1 mmol/l
ethanol), the solvents for BAL or BAPSA, respectively.
The efficacy of the antidotes was tested by comparing
arsenic elimination in the bile and the effluent perfusate
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 during 40 min. of perfusion before and after addition of the
time(min)
respective compound, 0.1 and 0.7 mmol/l, respectively. The
Fig. 1. Arsenic elimination in the bile during 40 min. of perfusion
before and after addition of antidotes (0.1 mmol/l), in percent of
results, given in percent of total liver toxicant burden before
the total arsenic liver burden before perfusion. Experimental details perfusion, are shown in table 2. The biliary excretion during
see table 2. the experimental period is shown in fig. 1 and 2.
354 F. X. REICHL ET AL.

Table 2.
Cumulative arsenic elimination in the bile and effluent perfusate during 40 min. of perfusion before and after antidote addition (0.1 and
0.7 mmol/l), in percent of total liver burden before perfusion (= biliary portion+eMuent perfusate portion+liver arsenic content after the
experiment) (mean va1uesfS.E.M. in paranthesis). The total arsenic content of the livers was 508.8k 10.0 pg Adliver.
% Arsenic elimination in the bile and effluent perfusate
0.1 mmol/l antidote
DMSA DMPS BAL BAPSA control
Bile before antidote 2.8 2.8 3.3 2.5 3.1
(0.4) (0.4) (0.2) (0.3) (0.7)
with antidote 9.6 13.4 3.5 13.6 3.8
(1.4) (1.2) (0.1) (1.4) (0.8)
Perfusate before antidote 64.6 64.0 63.7 65.0 63.5
(1.7) (0.6) (4.8) (3.3) (2.1)
with antidote 73.7 74.1 71.2 74.2 71.0
(2.1) (1.9) (4.7) (2.0) (1.4)
0.7 mmol/l antidote
DMSA DMPS BAL BAPSA control
Bile before antidote 2.9 2.8 3.2 3.0 3.1
(0.5) (0.2) (0.6) (0.2) (0.7)
with antidote 12.5 15.1 3.3 16.3 3.8
(1.3) (2.0) (0.6) (0.4) (0.8)
Perfusate before antidote 65.1 63.6 61.1 62.3 63.5
(2.7) (1.4) (4.7) (4.6) (2.1)
with antidote 75.1 76.6 64.2 76.1 71.0
(3.4) (1.4) (4.6) . (2.8) (1.4)
Guinea pigs distributed at random in groups of six animals received As20,, 10 mg/kg subcutaneously at 9 a.m. as a single injection. One
hr after the injection, the animals were anaesthetized, the livers removed and perfused with oxygenated (95% 0 , / 5 % C 0 2 )Krebs-Henseleit
buffer with glucose (10 mmol/l). After a 40 min. control perfusion period the antidotes were added to the perfusate for 40 min.

Relative changes of arsenic elimination was calculated as ation of arsenic in bile where DMSA was less and BAPSA
difference between the cumulative arsenic elimination 40 was most effective. In both cases no increase of effect was
min. before and 40 min. after antidote addition compared observed by the increase in the dose from 0.1 to 0.7 mmoll
to the difference in controls (fig. 3 and 4). 1.
DMPS, DMSA, and BAPSA caused an increase in elimin- A significant decrease of arsenic elimination in the bile
and perfusate was observed with BAL (0.7 mmol/l), com-
pared to controls. The bile flow in the controls was 2.9 It 0.3
p1 xmin.-' x g-' liver (meanfS.E.M.) during the 80 min.
% of change
OM perfusion period. After addition of 0.1 or 0.7 mmol/l
30 effluentperfusate DMPS, DMSA, BAPSA and 0.1 mmol/l BAL no significant
p < 0.05
change in the bile flow was observed compared to controls.
A decrease to 20% was found after 0.7 mmol/l BAL ad-
dition.
The bile/perfusate concentration ratio after 40 min. and
15 80 min. of perfusion is shown in table 3.

10
Discussion
Arsenic was eliminated via bile and effluent perfusate in the
controls.
Although more arsenic was found in the perfusate, the
DMSA DMPS BAL BAPSA
control high bile/perfusate concentration ratio (see table 3) of 40
mwoiaatnunoyI
(16 pmol As/l in the bile versus 0.4 pmol As/l in the perfu-
Fig. 3. Percent difference between the cumulative arsenic elimination sate) in control livers indicates that Some active processes
40 min. before and 40 min. after antidote addition (0.1 mmol/l) involved in biliary arsenic elimination.
compared to the difference in controls. (*) significantly different to
may
controls (PI 0.05). Following values were also significantly different The total arsenic excretion was increased following per-
to each other, bile: DMSA:DMPS; DMSA:BAL; DMSA:BDSA; fusion with DMSA, DMPS, or BAPSA, 0.1 or 0.7 mmol/l
DMPS:BAL; BALBAPSA. Experimental details see table 2. each, compared to controls.
 ANTIDOTES ON BILIARY ARSENIC EXCRETION 355

% of change tive arsenic excretion via bile was observed after addition
0bile of BAPSA (0.7 mmol/l).
effluent perfusate The finding that the bile/perfusate concentration ratio
)r "COO5 increases after addition of antidotes (exception BAL) sug-
gests a transport mechanism for antidotes. In the case of
20 DMPS even the entering of hepatocytes may be an anion
carrier energy dependent process, as was described for
15 * * ,T erythrocytes by Wildenauer et al. (1 982).
The pattern of arsenic elimination of molecules via bile
depends on its ionisation state, lipid solubility and molecular

0
' L4mL-m
5

0
weight (Goresky & Schwab 1988). Non-polar drugs with a
molecular weight lower than 400 50 will not be readily
excreted with the bile in guinea pigs (Abou El Makarem et
al. 1967). Essentially this is true also in other species
(Millburn et al. 1967a & b).
However, the molecular weight of all investigated anti-
Fig. 4. Percent difference between the cumulative arsenic elimination
40 min. before and 40 min. after antidote addition (0.7 mmol/l) dotes was lower than 400, so it seems that in biliary arsenic
compared to the difference in controls. (*) significantly different to excretion the molecular weight is only of minor significance.
controls (P10.05). Following values were also significantly different Ionized hydrophilic substances are excreted mainly by
to each other, bile: DMSA:BAL; DMSA:BAPSA; DMPS:BAL; bile (Goresky & Schwab 1988). In agreement with this, the
BAL:BAPSA, effluent perfusate: DMSA:BAL; DMPS:BAL;
BALBAPSA. Experimental details see table 2.
hydrophilic and at pH 7.4 completely ionized antidotes
DMPS and DMSA (0.1 and 0.7 mmol/l) increased the
biliary arsenic excretion compared to controls. In the case
of the lipophilic, non-ionized BAL (0.1 and 0.7 mmol/l) at
The percent difference between the cumulative arsenic pH 7.4, with a molecular weight lower than 400 the biliary
elimination 40 min. before and 40 min. after antidote ad- excretion of arsenic was not increased compared to controls.
dition was increased from 0.7% in controls of total arsenic The high biliary excretion of BAPSA (0.1 and 0.7 mmol/
liver burden to 13.3% with BAPSA (0.7 mmolll). This mo- 1) cannot be explained by the hydrophilic state nor by the
bilisation was increased although a great portion of the molecular weight of the compound. At physiological pH
easily available arsenic was eliminated during 40 min. pre- the lipophilic BAPSA exists in a non-ionized state and the
perfusion (see table 2). molecular weight is lower than 400. However, it cannot be
Arsenic was excreted only by perfusate after addition of excluded that adducts with BAPSA or other antidotes will
0.1 and 0.7 mmol/l BAL. The low biliary excretion of be formed by conjugation with other molecules, then ex-
arsenic could not be increased after the higher dose with ceeding the molecular weight level of 400 and resulting in
BAL. In contrast, the bile flow and the bile/perfusate con- increased biliary excretion.
centration ratio diminished to 20% and 22%, respectively, The results indicate that the treatment with DMSA,
compared to controls, indicating some 'toxic' effects after DMPS and BAPSA may cause a substantial shift to faecal
addition of 0.7 mmol/l BAL. elimination.
Against that, a significant increase in the biliary excretion Klaassen et al. (1974) described the enterohepatic circu-
was found after addition of DMPS, DMSA, and BAPSA lation of arsenic in rats, rabbits and dogs. Although some
(0.1 and 0.7 mmol/l), compared to controls. The most effec- enterohepatic circulation of complexed arsenic cannot be ex-
cluded, Maehashi & Murata (1986) have shown that dithiol
treatment increases the faecal excretion of arsenic and there-
Table 3. by can contribute to total arsenic elimination from the body.
Bileiperfusate concentration ratio. Mean values represent the ar-
A rapid arsenic elimination by addition of chelating
senic concentration in the bile versus effluent perfusate after 40 and agents which shift the toxicant elimination to the gut may
80 min. perfusion. have the advantage that the burden of the kidney and others
Bile/perfusate concentration ratio organs might become reduced after acute poisoning with
Antidote concentration As,O,.
0.1 mmol/l 0.7 mmol/l Acknowledgements
before after before after The skillful technical assistance of Mrs. Judith Friedrich,
antidote antidote antidote antidote Mrs. Renate Fischer, Mrs. Brigitte WeiD, Mrs. Petra Ebner,
Control 43 40 43 40 and Mr. Samir Islambouli is gratefully acknowledged.
BAL 40 38 38 30
DMSA 39 90 45 108
DMPS 41 129 39 148 References
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