| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Case Report |
1
Laboratoire de Biochimie, Pharmacotoxicologie et Analyse des Traces, Hôpital Edouard Herriot, Place D'Arsonval, 69437 Lyon 03, France.
2
Laboratoire de Chimie Analytique III, Faculté de
Pharmacie, Université Claude Bernard, 8 Avenue Rockefeller, 69373
Lyon 08, France.
3
Institut de Médecine Légale de Lyon, 12
Avenue Rockefeller, 69437 Lyon 03, France.
a Author for correspondence. Fax 33 4 7211 0617.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The arsenicals have been well known as poisonous agents since antiquity and have been used frequently in homicides and suicides. Lethal acute intoxication by arsenic has become rare, although arsenic is still sometimes used for suicide.
The acute toxicity of arsenic has been shown to vary according to the chemical species involved. The 50% lethal doses for oral administration to mice are as follows: 3 mg/kg for arsine; 14 mg/kg for arsenite [As(III)]; 20 mg/kg for arsenate [As(V)]; 700-1800 mg/kg for monomethylarsonic acid (MMA);1 700-2600 mg/kg for dimethylarsinic acid (DMA); and >10 000 mg/kg for arsenobetaine and arsenocholine (1). Therefore, the inorganic species of As(III) and As(V) are more toxic than their methylated forms MMA and DMA. Arsenobetaine and arsenocholine are nontoxic and are eliminated rapidly in the urine after absorption. The lethal dose of inorganic arsenic is 100–200 mg of arsenic trioxide for an adult individual, although people have survived larger doses (2)(3).
After its absorption, inorganic arsenic is partly methylated into MMA and DMA, which are excreted largely in the urine. One fraction of the remaining arsenic is eliminated unchanged in the urine; the second fraction is retained in the organism (2)(4)(5).
In healthy humans exposed to trace amounts of arsenic in their environment, the highest concentrations of arsenic are found in tissues rich in sulfhydryl groups, such as the skin, hair, and nails (2). Little is known about the total arsenic concentrations in the organs of unexposed individuals; the concentrations in blood and urine are 2.5 µg/L and 10–50 µg/L, respectively (2).
When larger doses of arsenic are ingested, the tissue distribution appears to change. Many studies have been performed in humans after fatal arsenic poisoning (6)(7)(8). The results of these studies showed widespread distribution of arsenic in all organs; the highest concentrations were in the liver and kidneys, which had, respectively, 10- and 3-fold higher concentrations than in the other organs (brain, cerebellum, lung, heart, pancreas, spleen, muscle, and skin). Although the distribution of total arsenic in tissues has been well studied in humans (6)(7)(8), few studies on the speciation of arsenic in animals have been reported in the literature, most of which focused on speciation in hepatic tissues following experimental acute intoxication (9)(10)(11)(12).
Here, we report a first case of arsenic speciation in human organs. The distribution of the arsenic species [As(III) and its metabolites] in each organ was studied, and the results were compared with those in the literature. Some of our results confirm known toxicological and metabolic mechanisms of inorganic arsenic in the human organism; others have led us to propose new mechanisms.
Arsenic speciation was performed using HPLC in the ion-pairing mode followed by arsenic determination by atomic absorption spectrometry (AAS).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mineralization was performed using nitric acid (Merck); deionized water and methanol (Carlo Erba) were used for arsenic extraction.
The mobile phase was prepared using deionized water; 400 mL/L tetrabutylammonium hydroxide (TBAH) in water (Fluka) was used as a counter anion, and orthophosphoric acid (normapur grade; Prolabo) was used to adjust the pH. Before use, mobile phase was filtered through a 0.2 µm cellulose acetate membrane (Millipore).
For graphite furnace AAS, 0.5 mL/L Triton X-100 (Merck) was used for sample dilution and 10 g/L Ni(NO3)2·6 H2O (Merck) was used as the matrix modifier. A 50 µg/L aqueous arsenic solution, prepared daily from a 1 g/L arsenic standard solution (Spex), was used to calibrate the AAS instrument.
We used a certified reference material: DORM-2 (dogfish muscle) from the National Research Council of Canada.
instrumentation
For separation of the arsenic compounds by HPLC, the
chromatographic system used included an isocratic pump (Shimadzu; model
LC-6A) and a Rheodyne valve fitted with a 20-µL sample loop.
Separation was performed in ion-pair mode on a reversed-phase silica-based octadecyl (C18) column resistant at pH 2–12 pH (SMT OD-5-100, 250 x 4.6 mm i.d.).
For the chromatographic optimization step, we used an ultraviolet detector (Shimadzu; model SPD-6A).
The determination of arsenic species in organ samples was performed using a SpectrAA Zeeman 220 (Varian) equipped with a pyrolytic graphite-coated graphite furnace, and a Zeeman-effect background corrector.
procedures
Specimens of organs (brain, cerebellum, heart, lungs, liver,
kidneys, pancreas, spleen, muscles, and skin) were collected, cut into
small pieces, and ground in a mixer (POLYTRON®).
Portions of the ground organs were divided in two aliquots, one for
total arsenic determination and the other for speciation. The
accurately weighed organ samples were heated for 6 h in an oven at
60 °C and completely dried for 3 h at 105 °C. The dried
samples were weighed and again put in the oven until the weight was
constant.
Total arsenic concentrations were quantified by AAS of dried powder samples of the organs after mineralization by nitric acid. The mean ( ± SD) total arsenic content determined for the reference material was 17.05 ± 1.6 µg/g (certified value, 18 ± 1.1 µg/g).
We extracted the arsenic species from the second powdered sample of each organ (400–500 mg dried weight) by adding 2 mL of 500 mL/L water–500 mL/L methanol (13)(14) and shaking the mixture for 20 min. The solution was centrifuged at 1950g and the remaining powder was extracted four more times, using the same procedure. The five extracts thus obtained were collected and evaporated. To obtain a concentrated arsenic solution from each residue (>800 µg/L), an appropriate volume of deionized water was added. This volume was determined after measurement of the total arsenic in the mineralizates. The solutions were shaken 20 min, centrifuged, and filtered through a 0.2 µm cellulose acetate membrane (Millipore).
For the chromatographic separation of arsenic species, we adapted our previously published method (15) for the separation of six arsenic species [As(III), As(V), DMA, MMA, arsenobetaine, and arsenocholine] in the ion-pairing mode. Optimization of the pH and counter ion (TBAH) concentration in the mobile phase was achieved for good separation of As(III), As(V), DMA, and MMA.
The study of the capacity factors of As(III), DMA, MMA, and As(V) as a function of the TBAH concentrations at neutral pH showed that the separation between DMA and the MMA is better at lower TBAH concentrations. The chosen concentration was 3 mmol/L. At this concentration, the influence of pH on the separation was studied, and pH 8 was selected as the optimal pH.
The selected working mobile phase contained 3 mmol/L TBAH in aqueous
solution at pH 8. The flow rate was 0.8 mL/min. The retention times for
the arsenic species, determined in a previous step by use of
concentrated calibrators detectable by ultraviolet absorption, were as
follows: As(III), 3.60 min; DMA, 6.60 min; MMA, 8.50 min; and As(V), 16
min. In a second step, the organ extracts were filtered through
a 0.2 µm Millipore membrane and injected into the column. Each
fraction of As(III), DMA, MMA, and As(V) was collected and concentrated
if necessary by evaporation at 40 °C. Each arsenic species in each
separated fraction was determined by AAS. The chromatogram of a liver
extract, with the arsenic species identified for the corresponding
fractions, is shown in Fig. 1
. Fractions (0.8 mL) were collected at 1-min intervals.
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. They show that arsenic is ubiquitous in all organs and that
the liver and kidneys contain the highest concentrations (147µg/g in
the liver and 26.6 µg/g in the kidneys). Other organs contain lower,
almost identical concentrations (8–12 µg/g). The total arsenic
concentration is lowest in the skin (2.9 µg/g).
|
extraction yields
The extraction efficiency depends on each organ. The results
presented in Table 2
show that the percentage of extraction by the methanol/water
mixture (calculated with mineralization as the reference method) is
between 71% and 98%.
|
determination of concentrations of arsenic species in organs
The concentrations of the arsenic species in different organs are
presented in Table 3
. In all tissues, As(III) was the main species found, comprising
75–85% of the total arsenic in most organs except in the cerebellum,
brain, and skin (47%, 53%, and 56%, respectively). The proportions
of the metabolites DMA and MMA are relatively small, and As(V)
concentrations are lower than the limit of quantification except in the
liver and kidneys.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) show that the arsenic tissular distribution is ubiquitous and that
the highest concentration is found in the liver, followed by the
kidneys. These findings confirm those reported in several studies of
fatal acute intoxication by inorganic arsenic in humans
(6)(8) and animals (9)(10)(11)(12). The concentration of arsenic in the liver and kidneys can be explained by the role of these organs in the detoxification (the liver is the site of inorganic arsenic methylation) and elimination of arsenic. It can also be noted the high concentrations found in muscle and heart (third and fourth highest arsenic concentrations after the liver and kidneys) tissue, which probably explains the fatal rhabdomyolysis observed with heart failure.
When we compare the concentration of total arsenic in all organs with
the concentration in the blood (Table 1
), we observe that the
concentrations in the organs are ~7- to 350-fold higher than in
blood. The blood sample studied was collected in the few hours
before death during the period of intense hemolysis.
The liver is the major target organ of inorganic arsenic, which is
explained by the affinity of As(III) toward vicinal dithiol in hepatic
cytosolic proteins. The concentration of total arsenic found in this
organ in our reported case was 5- to 50-fold higher than in most other
organs (Table 1
). Arsenic is distributed almost equally throughout most
of the other organs (8–12 µg).
The binding of arsenic in target organs was postulated as a first step of arsenic detoxification. Nevertheless, this binding is also responsible for the major intoxication symptoms: hepatic and renal failure and cardiovascular and neurological effects. Marafante et al. (9) performed an in vivo study in rabbits to determine the arsenic distribution in tissues. The results showed that 5 h after arsenite administration, 11.6%, 1.4%, and 1.7% of the dose was distributed in the liver, kidneys, and lung, respectively, most of which was bound to proteins. This binding occurred within 1 h of either arsenite or arsenate administration (16)(17). The binding of arsenic to tissue proteins has been proposed as an additional step or perhaps the first step in the detoxification of inorganic arsenic before methylation (18)(19).
arsenic speciation
Studies in the literature have demonstrated that, in
animals, As(III) is the major arsenic species present in tissues after
experimental intoxication with As(III) or As(V). These results
implicate As(III) as the main form of arsenic bound to tissues,
compared with As(V) and the methylated metabolites. In addition, these
studies showed that, in acute intoxication, arsenic cannot be
completely detoxified by the organism and binds to tissues. As(III) is
the main form of arsenic bound to hepatic tissue, compared with As(V)
and DMA (10)(16). Bogdan et al. (10)
reported that when hepatic cytosolic proteins were incubated with
inorganic arsenic, the amount of As(III) bound to these proteins was
13-fold greater than the amount of As(V) bound. In animals given
As(III), the majority of absorbed As(III) was found in the liver,
notably in the cytosol (50–65%) (20). This difference in
protein binding might be attributable to the affinity of As(III) for
sulfhydryl groups, especially vicinal dithiol (10).
These observations made in animal experiments appear to be confirmed in
humans by our results (Fig. 2
): we found As(III) to be three- to sixfold more concentrated
than other species (Table 3
, column 7). We also observed for the first
time a lower ratio of As(III) to other arsenic species in lipid-rich
organs (cerebellum and brain). This difference can be explained by the
increased affinity of the metabolites toward lipids compared with
As(III). Skin appears to be rich in the methylated metabolites,
although if has the lowest total arsenic content among all tissues. In
acute intoxication, compared with chronic intoxication, skin
concentrations are low (2)(6)(7)(8). The increased
concentration of arsenic metabolites in skin compared with other organs
remains unexplained. The ratio also equals unity in blood.
|
Our speciation results show the presence of four arsenic species: As(III), which is absorbed but not metabolized, MMA, DMA , and As(V).
These findings are in concordance with the knowledge of arsenic
metabolism in humans. Indeed, the methylation pathway involves the
formation of a monomethylated metabolite of As(III), which is either
rapidly methylated again into a dimethylated derivative or is
spontaneously oxidized into the As(V) form MMA (12). The
first methylation reaction is catalyzed by arsenite methyltransferase.
The transfer of the second methyl group is catalyzed by another enzyme,
MMA methyltransferase (20). The latter enzyme is sensitive
to inhibition by As(III) (11)(12). This
characteristic explains the increased concentrations of MMA in
biological media after acute intoxication
(3)(12)(21). As(V) is reduced to
As(III) by arsenate reductase before its methylation. In addition, a
small percentage of total arsenic is found in blood and urine as the
As(V) form and was shown to originate from in vivo oxidation of
absorbed As(III); the mechanism of this oxidation, however, is unknown
(2)(21). When viewed collectively, studies of
the transformations of ingested inorganic species in humans and mammals
(2)(11)(12)(21)(22) lead to the postulation of a general mechanistic
sequence: As(V) 
As(III) 
MMA DMA.
The methylated metabolites (MMA and DMA) show a very weak affinity for tissues; therefore, their elimination in bile, blood, and urine is rapid (2)(10)(23). Many studies have shown that MMA is the major metabolite in acute intoxication, whereas DMA is the major metabolite in chronic exposition (2)(23)(24)(25).
The MMA/DMA ratios in the tissues in our study (Table 3
, column 8)
indicate that MMA is 2.5- to 3-fold more concentrated than DMA, which
is characteristic of acute intoxication
(3)(12)(21). The much higher
concentrations of MMA compared with DMA can be explained by the
modification of the mechanism of methylation in acute intoxication. In
this case, MMA methyltransferase is inhibited by the high concentration
of As(III).
The amounts of the metabolites (Table 3
, column 6) are lower than
As(III); these species are eliminated rapidly in the bile and blood
after methylation because of their weak affinity for tissues.
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  D. J. Thomas, J. Li, S. B. Waters, W. Xing, B. M. Adair, Z. Drobna, V. Devesa, and M. Styblo Arsenic (+3 Oxidation State) Methyltransferase and the Methylation of Arsenicals Experimental Biology and Medicine, January 1, 2007; 232(1): 3 - 13. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R N Ratnaike Acute and chronic arsenic toxicity Postgrad. Med. J., July 1, 2003; 79(933): 391 - 396. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Unnikrishnan, J. P. Dutcher, N. Varshneya, R. Lucariello, M. Api, S. Garl, P. H. Wiernik, and S. Chiaramida Torsades de pointes in 3 patients with leukemia treated with arsenic trioxide Blood, March 1, 2001; 97(5): 1514 - 1516. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Gregus, A. Gyurasics, and I. Csanaky Biliary and Urinary Excretion of Inorganic Arsenic: Monomethylarsonous Acid as a Major Biliary Metabolite in Rats Toxicol. Sci., July 1, 2000; 56(1): 18 - 25. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
