Toxicology, 74 (1992) 69-76 69

Elsevier Scientific Publishers Ireland Ltd.

D-Penicillamine and prussian blue as antidotes

against thallium intoxication in rats

C a m i l o Rios a a n d A n t o n i o M o n r o y - N o y o l a b

aDepartamento de Neuroqulmica, Instituto Nacional de Neurolog]a y Neurocirugla, SSA and

bDepartamento de Toxicologia e Higiene Industrial, Escuela de Salud Pitblica, Instituto Nacional de Salud
Phblica, SSA (Mbxico)

(Received March 5th, 1992; accepted May 8th, 1992)

Summary

I>-penicillamine (DP) and prussian blue (PB), given alone and in combination, were evaluated in rats
as treatments against acute thaliotoxicosis. Animals were poisoned by intraperitoneal (i.p.) injection of
thallium(I) acetate at different doses (16, 30, 40, 50 and 70 mg/kg). Later (24 h), treatments were
administered until day 5, as follows: I>penicillamine (DP), 25 mg/kg, i.p. route, twice daily; prussian blue
(PB), 50 mg/kg, oral route, twice daily. LDs0 values were estimated for each treatment with the following
results: control, 32 mg/kg; DP, 27 mg/kg; PB, 42 mg/kg; PB + DP, 64 mg/kg. Thallium content was
analyzed in six body organs and eight brain regions after treatments. PB administration induced signifi-
cant elimination of thallium from all tissues. DP treatment diminished thallium content in body organs,
but increased it in brain regions, indicating a redistributive effect of DP. DP + PB treatment decreased
thallium content in all body organs and brain regions. Renal thallium content in the DP + PB group was
significantly lower than that of PB alone group, suggesting accelerated urinary excretion of thallium as
a result of DP action. Results indicate that DP administered alone may be dangerous because of its
redistributive effect, but given in combination with PB may be useful as treatment against thallium
poisoning.

Key words: Thallium; Thallium antidotes; Brain; Prussian blue; D-penicillamine

Introduction

Thallium salts are very toxic to man [1]. Most cases of human thallotoxicosis
occur after acute ingestion of the poison as sulphate, acetate or malate [2,3], either
accidentally or intentionally [4]. Toxic symptoms develop within a few days after
thallium intake, as gastrointestinal manifestations and nervous system dysfunction,
including ataxia, motor paralysis, and coma [5]. The use of antidotal treatment
against acute thallium intoxication is stressed because of the severity of the sym-
ptoms and the high lethality of the poison [6]. Prussian blue [KFe(Fe(CN)6)] (PB)
is orally administered as antidotal treatment in thallium intoxication cases. It has

Correspondence to: Camilo Rios, Departamento de Neuroquimica, Instituto Nacional de Neurologia y

Neurocirugia, Insurgentes Sur No. 3877, M~xico 14269, D.F.

0300-483X/92/$05.00
© 1992 Elsevier Scientific Publishers Ireland Ltd.
Printed and Published in Ireland
70

been demonstrated to be useful to accelerate tissue thallium elimination from its sites
of action (liver, kidney, brain), both in experimental and clinical studies [7];
therefore, it is considered the drug of choice against thallotoxicosis [8]. Chelating
agents have also been tested as antidotes, but their beneficial effects are doubtful
since they can cause redistribution of thallium from inactive depots to the target
organs such as brain, leading to an exacerbation of the symptoms [9]. D-penicil-
lamine (DP) is currently used as chelator of copper, lead and mercury in human cases
of intoxication [10], and has been also tested in clinical studies as therapy against
thallium poisoning [11,12]. No experimental evaluation of DP has been carried out
to date which provides a rationale for its use in human thallotoxicosis.
In the present work, we evaluate and compare the efficacy of PB and DP against
acute thallium intoxication in rats to test the possible therapeutic utility of these
compounds given alone or in combination.

Materials and Methods

Reagents
Thallium(I) acetate, D-penicillamine, Tween-80 and atomic absorption stand-
ard for thallium, were obtained from Sigma Chemical Co., Inc. (Milwaukee, WI).
All other reagents were from Merck (M~xico).

Animals
Male Wistar rats, NIH strain, weighing 200-250 g were used in all experiments.
They were fed a standard chow diet (Purina chow) and had free access to water.
Room darkness was maintained between 19:00 and 07:00 h, room temperature at
25°C and relative humidity at 40%.

Lethal dose (LDso) estimations

To obtain estimations of LDs0 values in the groups of rats with antidotal
treatments, 200 animals were intoxicated by i.p. injection of - 1 ml of aqueous
thallium(I) acetate solution at the following doses: 20, 30, 40, 50 and 70 mg/kg. These
values were chosen on the basis of the reported LDs0 for rodents (30 mg/kg) [13].
After (24 h) thallium injection, antidotal treatments on animals were begun as
described below. Ten animals were allocated to each group. The number of dead
animals in the different groups was recorded during the 4 days of treatment and the
day after treatment. For each group, LDs0 values were calculated using probit
analysis. Confidence intervals at the 95% level for LDs0 values were obtained as
described elsewhere [14].

Antidotal treatments
For antidotal treatments, rats were injected i.p. with variable doses of thallium
acetate (see the previous section) and randomly divided after thallium intoxication
into four groups of ten rats per dose each: Group C, control animals, treated with
vehicle; Group DP, animals injected i.p. with - 1 ml of D-penicillamine (dose = 25
mg/kg) dissolved in saline solution, twice daily; Group PB, animals orally treated
with - 1 ml of prussian blue (dose = 50 mg/kg) suspension in 1% Tween-80, twice
 71

daily; Group PB + DP, animals given DP and PB at doses and through the routes
already described. All treatments were begun 24 h after thallium intoxication and
maintained for 4 days (see treatments's administration, Table I). The dose of PB
used was previously reported to effectively induce decorporation of the metal [7].
Doses of 50 and 100 mg/kg of DP were also tested with results of mortality similar
to those described in the case of 25 mg/kg dose; therefore, the last dose was main-
tained throughout the experiments. Drugs were administered between 10:00 h and
12:00 h and between 18:00 h and 20:00 h.

Thallium analysis
To assess thallium decorporation and possible redistribution as a result of
treatments, the content of metal was analyzed in eight brain regions (cortex,
cerebellum, hippocampus, corpus striatum, hypothalamus, thalamus, midbrain and
pons), six body organs (liver, heart, kidney, lung, testis and spleen) and blood, using
a graphite furnace atomic absorption spectrophotometric method previously
described by our group [15]. In these experiments, we used a thallium dose of 16
mg/kg, which is one-half of the LDs0 calculated in the previously described experi-
ments. Animals were sacrificed for tissue thallium analysis 24 h after the last
administration of antidotes. Blood samples ( - 0 . 5 ml) were withdrawn by cardiac
puncture 5 days after thallium administration.

Statistics
Results of tissue thallium analysis were statistically analyzed using one-way
analysis of variance followed by Tukey's test for multiple comparisons [16].

Results

LD5o estimation
The effect of antidotal treatments on thallium LDs0 is shown in Table II. A s
expected, we found that PB protected rats against thallium lethality (LDs0 = 32,
control group, vs. LDs0 = 42, PB group). DP slightly enhanced thallium toxicity
(LDs0 = 32, control group, vs. LDs0 = 27, DP group), indicating that administered
alone, DP was not protective to animals. Surprisingly, DP given in combination with
PB resulted in a significantly higher protection against thallium poisoning than that

TABLE I

ANTIDOTAL TREATMENT ADMINISTRATION

Doses and routes of administration are described in Materials and methods.

Day 1 Days 2-5 Day 6

Thallium Antidotal treatments Tissue

administration twice daily sampling
72

TABLE II

THALLIUM L D ~ ESTIMATION IN THE A N T I D O T A L T R E A T M E N T GROUPS

Treatment LDs0 value (mg/kg) 95% Confidence interval

Control 32 (30, 35)

D-Penicillamine (DP) 27 (26, 29)
Prussian blue (PB) 42 (40, 46)
DP + PB 64 (61, 69)

exerted by PB alone (LD50 = 42, PB group, vs. LD50 = 64, PB + DP group),

suggesting a synergistic effect between DP and PB.

Thallium content in body organs

Thallium content in body organs is presented in Fig. 1. As clearly shown, PB treat-

80

70

60-

~' 50

4o

_E
~. 3 0 -
i-

4-
20

I0

All AllCOEF A|¢

JAle

DP PB DP - pll

Antidotal treatment

Fig. 1. Thallium content in body organs, Results are mean + 1 SEM of n = 10 experiments. C, control
group; DP, D-penicillamine group; PB, prussian blue group; DP-PB, combined DP + PB group; A, liver;
B, lung; C, spleen; D, heart; E, testis; F, kidney. Statistically different from respective control organ **
(P < 0.01), * (P < 0.05). ++All organ levels statistically different from respective control and DP group
organs P < 0.01. All organ levels statistically different from respective control and DP group organ levels
and kidney levels statistically different from respective PB group organ levels, P < 0.01.
 73

ment induced significant decorporation of the metal from all tissular depots studied,
perhaps as a result of increased thallium excretion through entero-hepatic route [18].
This accelerated decorporation may explain the higher than control LDs0 observed
in PB-treated rats. DP treatment induced a slight decrease in thallium content as
compared with levels in the control group, significant in all organs except liver,
indicating removal of the metal to other tissues (as brain) or accelerated excretion.
Thallium content in all body organs of rats after PB + DP treatment was
significantly diminished as compared both with controls and DP-treated animals.
Renal content of thallium in DP + PB group was significantly lower than that of PB
group (again, see Fig. 1), suggesting a DP-induced increase of kidney thallium
excretion in addition to the decorporative effect of PB.

Blood thallium levels

Levels of thallium in blood of rats 5 days after intoxication, presented in Fig. 2,
show a statistically significant decrease of thallium in the systemic circulation after
PB treatment as compared with control group levels. This result, together with that
of tissue thallium reduction previously described, indicates that PB can reduce body
burdens of thallium significantly. DP treatment, on the other hand, was unable to

..J

oo
E "r T

E
-X
I- ÷+
e- "1"
l-

"0
0
0 u

C~D

0 c DP PB DP-PB

Antidotal Treatment
Fig. 2. Thallium in blood levels. Results are mean + 1 SEM o f n = 10 experiments. C, control group; DP,
D-penicillamine group; PB, prussian blue group; DP-PB, combined DP + PB group. *Statistically dif-
ferent from control and DP group levels (P < 0.05). ++Statistically different from control and DP group
levels (P < 0.01).
74

reduce circulating thallium as shown in Fig. 2. C o m b i n e d D P + PB treatment also

diminished blood thallium contents significantly as c o m p a r e d with those o f controls,
but not as c o m p a r e d with those o f PB-treated animals.

Thallium content in brain regions

Brain regional thallium content is shown in Fig. 3. PB administration significantly
decreased brain thallium in all regions studied as c o m p a r e d with the control levels.
Brain regional thallium content in DP-treated animals was found slightly increased
in all regions as c o m p a r e d with controls, reaching significance in corpus striatum,
midbrain and hypothalamus, indicating that D P induced redistribution o f thallium
from other tissues to these brain regions. Redistribution m a y be dangerous to
animals and is suspected to be responsible for the lower LDs0 value observed in
DP-treated animals.
D P + PB group showed significantly lower than control levels o f thallium in all
brain regions. C o m p a r e d with PB group, D P + PB group showed a slight non-
significant increase in brain regional thallium content.

Discussion and conclusions

Early investigations on thallium antidotal treatment conducted by Kamerbeek et

al. demonstrated that PB is able to induce thallium decorporation in the thallium-
intoxicated rat [7]. These results, however, were not related to the ability o f PB to

20

~. f5
-?
~o
L
U

i--

1234 34 678 I 2 3 4 5 6 7 8 I 2 3 4 5 6 7
c DP PB DP PB
-

Antidotal T r e a t m e n t

Fig. 3. Thallium content in brain regions. Results are mean + 1 SEM of n = 10 independent experiments.
C, control group; DP, D-penicillamine group; PB, prussian blue group; DP-PB, combined DP + PB
group; 1, cerebellum; 2, frontal cortex; 3, pons; 4, thalamus; 5, midbrain; 6, cortex; 7, hippocampus; 8,
hypothalamus. Statistically different from respective control region ** P < 0.01, * P < 0.05. ++All
region levels statistically different from respective control and DP group region levels, P < 0.01.
 75

prevent thallium-induced mortality of rats. Our study showed for the first time that,
after 4 days of PB treatment, rats were effectively protected against thallium lethal-
ity. This protection seems to be the result of thallium-accelerated excretion through
feces, after PB interruption of the entero-hepatic recycling of the metal [17]. In
agreement with those early reports, our results indicate that PB treatment resulted
in significant removal of thallium from depots to excretion without dangerous
redistributive effects.
Thallium poisoning in man has been treated with chelating agents, like sodium
diethyldithiocarbamate (NaDDC) with which thallous ion forms a lipophilic com-
pound [18]. Even when this treatment increased urinary excretion of thallium, side
effects were observed, e.g. central nervous system dysfunction (for a review, see Ref.
8). Kamerbeek et al. demonstrated that NaDDC treatment in rats caused redistri-
bution of thallium to the brain, producing serious side effects [9]. In our study, D-
penicillamine treatment was able to induce a small but significant decrease of
thallium content in all organs studied except brain, in which DP increased thallium
levels through a mechanism perhaps similar to that of NaDDC. Our results indicate
that DP administered alone produce redistribution and this may be an important
factor in potentiating TI lethality in DP-treated rats. However, DP-induced
redistribution, at the dose employed, seems to be less potent than that produced by
NaDDC administration. According to Kamerbeek et al., a single NaDDC injection
doubled the brain thallium concentration in thallium-poisoned rats, as compared
with NaDDC-untreated controls [9]. These results suggest that thallium-DP adduct
may be less lipophilic than thallium-DDC complex.
It has been reported that initial D-penicillamine therapy in Wilson's disease, a
condition caused by excessive free copper in the brain, can result in worsening of
the associated neurologic syndrome [19], suggesting a redistributive effect of
D-penicillamine on copper content similar to that observed for thallium in this
work. In addition, cadmium-induced lethality in the rat is significantly increased by
D-penicillamine administration [20], suggesting similar mechanisms of enhanced
toxicity for both metals in the presence of DP.
Significant redistribution of thallium was no longer observed when PB and DP
were co-administered. This may be the result of a diminished amount of thallium
available for redistribution, after the accelerated excretion induced by PB.
Mechanisms of tissue protection by DP other than metal decorporation have been
proposed and might be applied to explain the protective effect of the compound
against thallium poisoning when administered together with a decorporating anti-
dote such as PB. These mechanisms include: (i) non-toxic metal-DP complex
formation, yielding harmless, bound metal [21] and (ii) metallothionein synthesis
induction, which may decrease free metal content [22]. These possibilities remain to
be tested in future experiments.

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76

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