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Arsenite medicinal use, metabolism,

pharmacokinetics and monitoring in human
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Article in Biochimie July 2009
Impact Factor: 2.96 DOI: 10.1016/j.biochi.2009.06.003 Source: PubMed

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Biochimie xxx (2009) 18

Contents lists available at ScienceDirect

Biochimie
journal homepage: www.elsevier.com/locate/biochi

I.

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E. Curis, P. Deschamps, S. Benazeth

Laboratoire de Biomathematiques, EA 2498, Departement de Sante Publique et Biostatistiques, Plateau iB2, Faculte de Pharmacie, Universite Paris Descartes, Paris, France

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 6 March 2009
Accepted 4 June 2009
Available online xxx

Acute promyelocytic leukaemia (APL) is a distinctive subtype of acute myeloid leukaemias. Even through
this human disease can be treated by the intravenous administration of all-trans retinoic acid (ATRA),
25% of patients typically relapse after the rst treatment. In this context, the intravenous administration
of APL patients with an aqueous solution of arsenic trioxide has also been demonstrated to be successful
despite the established mammalian toxicity of this arsenic compound. Accordingly, the administration of
a therapeutic dose of arsenic trioxide has resulted in an improved patient survival in both relapsing as
well newly diagnosed APL patients.
We present here a mini-review of the medicinal use of arsenite, its mammalian metabolism (with an
emphasis on biomethylation pathways), its elimination and pharmacokinetics and the novel application
of hair analysis as a biomonitoring material. This mini-review also introduces our own results on the
analysis of hair of patients receiving arsenic trioxide therapy.
In this work, instead of quantifying arsenic content in bulk hair, we performed longitudinal analysis in
order to use hair as a marker of arsenic exposure correlated to a time scale. Taking into account the hair
growth rate, the longitudinal analysis of hair is demonstrated to provide a chronological record of the
treatment of patients with arsenic trioxide. The small quantity of material to be analysed required the use
of Synchrotron radiation based X-ray uorescence (SXRF) spectroscopy. The hair arsenic content was well
correlated with the clinical background of patients and reected the intake of arsenic trioxide. In
particular, the onset of arsenic trioxide therapy and interruptions during therapy were reected by total
arsenic content, which suggested rapid elimination.
Another type of experiment, micro-XRF cartography on thin hair slices, allowed us to obtain distribution maps of arsenic, which demonstrated that arsenic is located at the periphery of hair. Micro-XANES
spectra recorded at the periphery of hair, suggest that inorganic arsenic is incorporated in hair in its
trivalent oxidation state, in agreement with previous results.
2009 Published by Elsevier Masson SAS.

Keywords:
Hair
Arsenic
Speciation
Synchrotron induced X-ray uorescence
Acute promyelocytic leukaemia

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1. Introduction

Leukaemias account for over 3% of total cancer mortality in

Europe and North America. [1] Acute promyelocytic leukaemia
(APL) is a distinctive subtype of acute myeloid leukaemias (AML),
which represents approximately 1015% of adult AML.[2] Since the
late 80s, APL is treated by a combination of standard chemotherapy
with all-trans retinoic acid (ATRA). Since the late 90s, however,
injectable aqueous arsenic trioxide [36] has been also successfully
used for the treatment of relapsing APL patients and, more recently,
in newly diagnosed patients.

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Nicolis*,

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Arsenite medicinal use, metabolism, pharmacokinetics and monitoring

in human hair

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Mini-review

* Corresponding author. Tel.: 33 153739778; fax: 33 153739777.

E-mail address: ioannis.nicolis@parisdescartes.fr (I. Nicolis).

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Our group, in collaboration with researchers located in several

Paris hospitals, works on the stability and bioavailability of nutritional supplements and medicinal drugs, such as arsenite to treat
leukaemia. Using EXAFS (Extended X-Ray Absorption Fine Structure) and XANES (X-ray Absorption Near Edge Structure)
spectroscopies, we previously established that arsenious acid
[As(OH)3] is the only detectable molecular form present in the
injectable solution of arsenic trioxide. In the aim to study the
assimilation and elimination of the drug after administration, we
obtained hairs of two patients having received the arsenic therapy.
As the hair stores the trace elements from blood [7] arsenic exposure can be followed along the hair before, during and after the
treatment period.
In a rst step of the study, we performed Synchrotron based
X-ray uorescence (SXRF) experiments in the LURE French
synchrotron facility, following thus the arsenic content along hairs

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0300-9084/$ see front matter 2009 Published by Elsevier Masson SAS.

doi:10.1016/j.biochi.2009.06.003

Please cite this article in press as: I. Nicolis et al., Arsenite medicinal use, metabolism, pharmacokinetics and monitoring in human hair, Biochimie (2009), doi:10.1016/j.biochi.2009.06.003

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I. Nicolis et al. / Biochimie xxx (2009) 18

The mechanism of action is dual depending on dose. At high

concentrations it implies induced apoptosis in leukaemic cells
depending on the activity of the enzymes that regulate cellular
H2O2 content [3,23,28]. At lower doses it induces partial differentiation of the myeloid cells.[29] Degradation of the PML-RARa
chimeric protein could contribute to both effects.[3032]
Treatment of relapsing APL with a 1 mg/mL arsenic trioxide
intravenously administered solution (Trisenox) has been
approved by the FDA in September 2000. All studies indicate an
improved patient survival and increased complete remissions for
both relapsing and newly diagnosed patients in particular in
combination with ATRA. Numerous studies in the last decade have
conrmed arsenic trioxide as a successful treatment for
APL.[20,3335] Arsenic trioxide and organic arsenic compounds are
tested also for other cancers as well [3639] and trials are performed to adjust treatment to different age groups [40,41].

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2. Arsenic and cancer treatment

3. Arsenic metabolism

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Arsenic is a well-known naturally occurring metalloid. The rst

written reference of arsenics medical use goes back to the 5th
century B.C.: Hippocrates of Cos quotes, among other ointments for
wound healing, one containing a mixture of two sulphur arsenicals,
orpiment and realgar.[10] The remedy is referenced as Caria
medication, which probably indicates that the formulation is even
older. However, it is not before Celsus on the 1st century of our era
that we found an explicit indication for arsenic used against solid
tumors.[11] Since then, arsenic has been empirically used for
various diseases, in particular after the introduction of the wellknown Fowler solution in 1788, a potassium bicarbonate-based
solution of arsenic trioxide. In 1878 the rst report of white blood
cell decrease after administration of the Fowler solution has been
published, particularly including a chronic myelogenous leukemia
(CML) patient.[12] Consequently, arsenic trioxide based therapy
became the main antileukemic treatment until, rst, the advent of
radiotherapy in 1903[13] and, later, the development of cytotoxic
chemotherapy in the second half of the 20th century. Despite
reports of successful treatments during the 30s [14], serious
evidence about the chronic poisoning of treated patients [15] put
an end to arsenic medication against CML.
Acute promyelocytic leukaemia (APL) is characterised by a t(15;
17) (q22; q21) chromosome translocation (more than 95% of
patients) leading to the fusion of the RARa and PML genes.[16] The
PML-RARa chimeric protein inhibits normal myeloid differentiation
leading to an accumulation of the leukaemic cells at the promyelocytic stage of development, severe coagulopathy and high early
mortality. In addition the PML-RARa prevents apoptosis. All-trans
retinoic acid (ATRA) degrades and cleaves the PML-RARa oncoprotein, leading thus to a signicant increase of patient
survival.[1720] Nevertheless 25% of patients have a relapse after
the rst treatment, leaving only bone marrow transplantation as an
option, although only for the younger relapsed patients.[2]
From the beginning of the 80s a Chinese group reported
successful use against APL of a mixture (referenced as Ai-lin I or
Ailing-1) containing arsenic and mercury in low doses [21,22]. This
report was followed by a detailed study of a pure arsenic trioxide
solution in intravenous infusion yielding complete remission for 9
among 10 patients.[3,4,23] Because of the well-established arsenic
toxicity, caution was initially suggested.[24] A subsequent US study
reported complete remission for 11 among 12 patients suffering
APL after arsenic trioxide administration.[2] This pilot study was
followed by a multicenter study on 40 relapsing APL patients, of
which 85% achieved complete remission.[25] The safety prole of
the drug is favourable at therapeutical doses and adverse events are
reversible.[6,26,27]

Arsenic metabolism is a subject of numerous studies as it

proceeds via a particularly complex pathway and has extensively
been studied in view of its toxicity.[4244]
In the past it was generally admitted that the methylation
pathway is a detoxication process as the methylated arsenic
compounds were considered less toxic than the inorganic arsenicals.[45,46] However, in the last decades many published studies
question this view [4749]. Indeed, evidence about the cytotoxicity
of methylated compounds appeared [50]. In particular, although
methylated arsenic (V) compounds are less toxic than inorganic
ones [51], the trivalent intermediates formed during the methylation process are much more toxic.[49,5153] Obviously the details
of the methylation pathway are highly relevant for an understanding of arsenic toxicity as well as of bioavailability in a therapeutic context.
While it has long been accepted that arsenic is metabolized via
a succession of oxidative methylation and reduction steps leading
from inorganic trivalent arsenic to pentavalent dimethylarsinic
acid, recent studies proposed an alternative reductive methylation
pathway. We summarize hereafter the main characteristics of those
pathways.

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of patients rst with a coarse 5 mm step [8,9] and after with a ner
3 mm step. These preliminary LURE experiments have been rened
in the ESRF European synchrotron facility, conrming correlations
between the therapy protocol and the hair content.
We present here a mini-review on arsenite medicinal use,
metabolism, pharmacokinetics and dosage in hair, followed by our
own results on hair analysis. Three types of measurements are
presented: ne step longitudinal hair X-ray uorescence spectroscopy, transversal hair cartography by spatially resolved X-ray
uorescence spectroscopy, and micro-XANES around the arsenic Kedge (11 867 eV) on thin hair sections. Focalisation and intensity
achieved with synchrotron X-ray sources allows the analysis at the
micro scale. With these three experimental techniques we obtain
kinetic information on arsenic inclusion along hair, trace element
distribution perpendicularly to the hair axis and chemical speciation of arsenic incorporated in hair.

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3.1. Oxidative methylation pathway (Fig. 1)

The pioneering work of Challenger on biological methylation
[54], provided the rst detailed view of the oxidative methylation
pathway. The main characteristic of this pathway is that only
trivalent arsenic compounds can be biomethylated, while

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iAsV
arsenate

1.20.4.1
arsenate reductase
or
2.4.2.1
purine nucleoside phosphorylase
1.20.4.2
methylarsonate
reductase

MMAIII
monomethylarsonous acid

iAsIII
arsenite
2.1.1.137
arsenite
methyltransferase

dimethylarsinic acid
(cacodylic acid)

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MMAV

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DMAIII
dimethylarsinate
reductase

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monomethylarsonic acid

2.1.1.137
arsenite
methyltransferase

DMAV

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dimethylarsinous acid

Fig. 1. Arsenic oxidative methylation pathway.

Please cite this article in press as: I. Nicolis et al., Arsenite medicinal use, metabolism, pharmacokinetics and monitoring in human hair, Biochimie (2009), doi:10.1016/j.biochi.2009.06.003

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arsenate

GSH

arsenite

AIIITG
arsenic trigluthathione
2.1.1.137
arsenite
methyltransferase

MMAV

MMAIII

monomethylarsonic
acid

monomethylarsonous
acid

GSH

DMAV
dimethylarsinic acid
(cacodylic acid)

DMAIII

dimethylarsinous acid

GSH

An alternative metabolic pathway has been recently proposed,

which proceeds by non-oxidative methylation, cancelling the need
for intermediate reduction steps.[71,72] Via this scheme, arsenate
needs still to be reduced to arsenite as exposed above but instead of
undergo oxidative methylation it forms an arsenic triglutathione
complex. This complex, substrate for arsenite methyltransferase,
can be methylated without oxidation directly to methylarsonic
diglutathione, which can be further methylated by the same arsenite methyltransferase to dimethylarsinic glutathione. At low
glutathione concentrations, both methylarsonic diglutathione and

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methyl arsenic
digluthathione

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2.1.1.137
arsenite
methyltransferase

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iAsIII

DMAIIIG
dimethyl arsenic
gluthathione

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3.2. Reductive methylation pathway (Fig. 2)

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1.20.4.1
arsenate
reductase

iAsV

Fig. 2. Arsenic reductive methylation pathway.

dimethylarsinic glutathione are hydrolysed to trivalent monomethylarsonous and dimethylarsinous acids, further oxidized by
H2O2 to monomethylarsonic and dimethylarsinic acids respectively
[73]. Interestingly, it is suggested that the glutathione complexes
are the arsenical compounds transported from the liver to the
blood stream and kidney and it has been found that both methylarsonic diglutathione and dimethylarsinic glutathione are more
stable than arsenic triglutathione.[74]

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pentavalent ones have to be reduced rst in order to undergo

further biomethylation. While Challenger suggested formaldehyde
as the methyl donor, it was later proved that S-adenosylmethionine
(SAM) is the methyl donor for the consecutive methylations.[55,56]
Several groups worked on the characterisation of the enzymes
involved in the biomethylation and reduction steps of the pathway
and two mechanisms are proposed.
The enzyme catalyzing the biomethylation has been identied
as arsenite methyltransferase (E.C. 2.1.1.137, also named Cyt19).[57]
It is a 375 amino acid cytoplasmic protein with an MW of 41 748 Da
coded by the gene AS3MT located in chromosom 10.[58] The same
enzyme can use as substrate either inorganic arsenite (AsIII) to
methylate it towards monomethylarsonic acid (AsV) or monomethylasonous acid (AsIII) to methylate it towards dimethylarsinic
acid (AsV).[47,59] The arsenic metabolism pathway shares many
common features among prokaryotes and eukaryotes [60]; homologues of Cyt19 protein, with a signicant conservation of ve
cysteine residues, have been found in a wide variety of species
attesting a well conserved function [61]. Nevertheless, some
species lack arsenite methyltransferase activity, mainly new world
animals with the noteworthy exception of chimpanzee, only old
world mammal not methylating arsenic [62]. It has been suggested
that this deciency is an evolutionary advantage for animals
exposed to trypanosomal diseases, as the absence of methylation
maintains chemotherapeutic levels of arsenite in the animals blood
and liver.[47]
The oxidative methylation scheme described above, requires
reduction of the pentavalent arsenic species in order to proceed.
Several glutathione dependent enzymes have been proposed as
putative reductases for these reductions. The inorganic pentavalent
arsenate is reduced in bacteria by an arsenate reductase (E.C.
1.20.4.1) while in humans a glyceraldehyde-3-phosphate dehydrogenase has been reported working in vitro as arsenate reductase.[63] A methylarsonate reductase (E.C. 1.20.4.2) has been
proven in vitro in rabbit and hamster liver extracts to reduce
monomethylarsonic acid to monomethylasonous acid [64,65] and
has been identied as a glutathione-S-transferase omega
(GSTO).[66] However, in GSTO knock out mice a reductase activity
remained, suggesting alternative pathways.[67] An other route for
reduction of arsenate to arsenite was identied, involving purine
nucleoside phosphorylase (E.C. 2.4.2.1) in the presence of dihydrolipoic acid. The hypothesis explaining this mechanism is that
since arsenate is chemically similar to phosphate, it can substitute
for phosphate, resulting in the formation of ribose-1-arsenate.[68]
Another proposition for the reduction steps of the pathway is that
both oxidative methylation and reduction activities are fused in the
same protein, arsenite methyltransferase. Indeed, it has been found
that in the presence of reducing agents, recombinant rat and
human arsenite methyltransferase could sustain the whole
pathway from inorganic arsenate to trimethylarsine oxide.[69] It
appears plausible that all these reduction mechanisms could
redundantly occur in vivo.[70]

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4. Arsenic pharmacokinetics
All pharmacokinetic studies agree on the rapid kinetics of
arsenic metabolism and rapid decrease of arsenic species in blood
after intravenous administration at the FDA approved dose of
0.15 mg As2O3/kg body wt. During the rst 24 h after administration trivalent inorganic arsenite is the main compound found in
urine, while pentavalent metabolites monomethylarsonic and
dimethylarsinic acids become the major urine arsenic species after
the rst 24 h, dimethylarsinic acid being generally the most
important one in percentage.[75,76] Only small amounts of
pentavalent inorganic arsenate are detected in urine. Because of
spontaneous oxidation of trivalent to pentavalent methylated
compounds, only recently the highly toxic monomethylarsonous
and dimethylarsinous acids have been detected in urine
samples.[75,7779] Contradictory results are published concerning
excretion routes. Urinary excretion is reported as a minor route for
elimination with 8% of daily dose by Shen et al.[4] but as a major
elimination route by Fukai et al.[76] which report 127% excretion of
the daily dose after repeated administrations. Other authors report
intermediate values ranging from 18%[80] to 65%[75] after intravenous administration or 4660% after oral ingestion [8185]. Oral
administration of As4S4 results in ca. 70% urinary excretion.[86] A
study of urinary excretion as a function of time on patients
receiving daily intravenous doses of arsenite reports a urinary
excretion of 20% on the rst day of therapy but maintained at 60%
after the rst week.[87] It is noteworthy that large variations are
reported among individuals concerning arsenic methylation which
probably affect toxicity and response to therapy.[88]
In samples collected from patients for three weeks after the last
administration of remission induction therapy, blood cells arsenic
content was measured 610 times higher than plasma levels.[89]
Pentavalent arsenic is found in blood only transiently at the end of
therapy and rapidly disappears. [40,89] In one study pentavalent
arsenic is observed at higher concentrations [87] but this is possibly
an artefact as analyses have been performed ve years after sample
collection (conserved at 20  C) and trivalent arsenic is known to
be oxidised to pentavalent even at 4  C after two months.[90]

Please cite this article in press as: I. Nicolis et al., Arsenite medicinal use, metabolism, pharmacokinetics and monitoring in human hair, Biochimie (2009), doi:10.1016/j.biochi.2009.06.003

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5. Synchrotron radiation applied to hair analysis for trace

monitoring and speciation

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5.1. Hair as a biomarker

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Hair has widely been used to assess content in a broad range of

substances, organic and inorganic, as well endogenous metabolites
or exogenous contaminants.[98] Most often, due to low concentrations and small sample quantities, analysis of several strands of
hairs was performed after homogenization and usually dissolution.
However, a unique property of hair is that incorporated elements
during its growth remain in place. Therefore, hair can be used to
retrace the history of exposure to a pollutant or the evolution of
a biomarker during time. In order to achieve such a past history
tracing, assay of hair composition has to be carried out along single
hair, the more distant points from the scalp being the older ones. Xray uorescence has successfully been used for such longitudinal
analysis in a case of fatal Hg exposure [99], to monitor zinc status
along time [100] or trace elements during pregnancy [101]. A wellknown subject is the controversy about Napoleon I arsenic hair
content which has extensively been studied by SXRF.[102] Microparticle induced X-ray emission (PIXE) is also well suited for such
analyses [103] In these studies segments of hair (1 or 2 mm long)
have been analysed.
However, in case a ner resolution is required, the problem
arising is the small mass of material to be analysed and only few
microanalytical techniques allow such resolution and sensitivity.
Micro-particle induced X-ray emission (PIXE) and synchrotron
induced X-ray uorescence (SXRF) are particularly suited for
measurements on small spots of biological samples because of the
focalisation and the intensity of the beam.[104] SXRF on hair has
been used both for assessing bioindicators as copper and zinc [105]
or external contaminants [102].

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The use of synchrotron radiation in hair study is not limited to

longitudinal analysis. Both SXRF [106] and PIXE [107,108] have been
used for cartography of metal distribution in hair sections, sometimes trying to distinguish endogenous from exogenous metal hair
content. SXRF has also been combined with Fourier transform
infrared microspectroscopy (FTIRM) in order to correlate metal and
protein distribution.[109]
Another question concerning metal content in hair is the
particular chemical form in which it is present. Most often extraction followed by chromatography coupled to mass spectrometry
techniques are applied.[79,110,111] However, X-ray absorption
spectroscopy (XAS) and particularly micro-XANES is a nondestructive technique that can differentiate many arsenic
compounds [112]. The advantage of this technique is that it allows
in situ determination without prior extraction and has been applied
to achieve arsenic speciation in hair [108].

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5.2. Arsenic monitoring, cartography and speciation in APL

patients hair

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Trivalent arsenic concentration of blood rapidly decreased after the

end of administration [87,89] falling below detection limits 10 h
after end of infusion [40] Mono-and di-methylated metabolites
appear in blood 6 h after arsenic infusion [87] and decreased more
slowly over few days, with dimethylarsinate being the major
metabolite after 4 days [89]. It is noteworthy that in another study
[76] monomethylarsonous acid has been reported as the major
metabolite after repeated administration indicating incomplete
methylation in liver. Except for one study [89], no accumulation of
inorganic arsenite is found in blood during daily repeated administration of arsenite [40,87] while trivalent methylated metabolites
continuously increase [89] to reach a steady state in some studies.
Finally thioarsenicals, in particular arsenic triglutathione and
methylarsonic diglutathione but not dimethylarsinic glutathione
thioarsenicals, are also detected in bile.[9194]
Elimination half times for inorganic arsenite and arsenate are
found from non-compartmental models to be 17 h and 18 h
respectively [87] after arsenite infusion. An elimination half life of
70 h is reported for As4S4 oral administration.[95] In a low dose
study (ca. half the FDA approved dose), pharmacokinetic analysis
yields a two phase process with half lifes of 1.4 and 9.4 h.[31] A noncompartmental analysis of total arsenic elimination has reported
a half life greater than 24 h, noting however that 10 h after the end
of the infusion inorganic arsenite was below detection limit thus,
the metabolites contribute to the half life extension.[40] A four
exponential model tted on total arsenic of only one individual
yields a small rst term with a 4 h half life, two major terms with
half lifes of 1.42 and 7.7 days and a minor fourth term with
a 44.1 days half life.[85] A variety of physiology based pharmacokinetics models are developed mainly to model oral ingestion after
environmental exposure [96,97].

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I. Nicolis et al. / Biochimie xxx (2009) 18

5.2.1. Materials and methods

Samples consist of human hair, provided for analysis by chemists
from the Pharmacie Centrale des Hopitaux de Paris. All measurements
were performed by microbeam synchrotron X-ray Fluorescence (mSXRF) on ID22 beamline at the ESRF synchrotron radiation facility.
All experiments have being performed under ambient temperature
and pressure conditions. X-ray uorescence spectra were excited
with a monochromatic beam of 14 keV energy using the Si [111]
double crystal monochromator and collected with a Si(Li) detector.
For kinetic experiments, three hairs of each of two patients were
mounted on a sample holder allowing 5 cm translation along hair
length. X-ray uorescence spectra, have being recorded along hair
with steps of 100 mm. Our previous coarse recordings [8] were used
to only record the arsenic containing portion of hair, that is from
10 mm to 45 mm from hair root for patient 1 and from hair root to
35 mm from it for patient 2.
For micro-XRF cartography and micro-XANES, transversal
human hair sections (20 mm thick) were cut with a microtome and
deposited on 4 mm polycarbonate lms. No glue was used, as
electrostatic attraction was sufcient to immobilize the hair slices.
The beam was focused to 1  3 mm2 with Kirkpatrick Baez double
mirror devices, which deliver 2.0  1011 ph/s at 14 keV. Stepsize for
cartography was 1  3 mm2.
Arsenic chemical speciation was investigated locally at the
points of hair with high arsenic content from m-XRF maps by
scanning around Arsenic absorption K-edge (11.863 keV) with
a 0.4 eV energy step. Scans were collected in the uorescence mode
and averaged in order to improve statistics. A series of As(III) and
As(V) inorganic and organic model compounds micro-XANES
spectra were recorded under the same conditions. XANES spectra
features are very well distinguished and they reect both oxidation
degree and ligand nature (sulfur or nitrogen/oxygen).[113]
Data were analysed using in house developed software
combining XAS analysis and image processing.[114]

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5.2.2. Results and discussion

Human hair grows at an average rate of 1.1  0.1 cm per month
(350 mm per day)[115,116], thus a recording of 1 cm of hair yields
the history of approximately a month of past exposure. The 100 mm
step we used corresponds to a sampling at a ca. 7 h interval. The
youngest hair being near the scalp, we go back in history moving
towards away from hair root. In Fig. 3 a double horizontal axis
indicates both distance from hair root and time before hair cut.
For both patients, upon beginning of therapy, we observed
a steep increase in arsenic content, which, in less than 24 h, reaches

Please cite this article in press as: I. Nicolis et al., Arsenite medicinal use, metabolism, pharmacokinetics and monitoring in human hair, Biochimie (2009), doi:10.1016/j.biochi.2009.06.003

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hair cuts of patient 1 on the beginning and the end of the arsenic
plateau and with hair cuts of patient 2 at the points with high
arsenic content (data not shown). This is in contrast to arsenic
distributions found in red squirrels fur living in an arsenic
contaminated environment where arsenic was found concentrated
in the centre of hair at the medulla region [108]. However, medulla
is rarely present in human hair and optical microscopy observation
of our samples did not reveal its presence. In previously published
human samples, arsenic was found mainly in the cortex area but,
depending on the authors, it is reported in the hair periphery [117]
for ingested arsenic or uniformly distributed for ingested arsenic
and more present in the periphery for external contamination.[118]
As we clearly found endogenously incorporated arsenic in hair
periphery, it appears that localisation of arsenic in hair section
cannot be used to distinguish internal or external arsenic content.
Micro-XANES spectra of high arsenic content hair spots (Fig. 5),
demonstrate that arsenic is under the As(III) oxidation state, i.e. as
administered and not oxidised to As(V) nor methylated as the
metabolites found in urine. We observe a 4 eV shift of the white line
between arsenite and arsenate in agreement with previously
published values.[112] This nding implies inclusion of As in hair
before oxidation, in accordance to our kinetic observations about

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more than half the maximum observed level. The differences

between the two patients correspond to their clinical background.
Indeed, patient 1 received a continuous administration of arsenic,
while patient 2 received arsenic for 40 non-continuous days in
a period of ca. two months, yielding an arsenic hair content pattern
of three cycles of high arsenic level separated by two periods of
lower level. For both patients the total hair length between initial
rise and nal decrease of arsenic content is ca. 2 cm, corresponding
to a little less than two months of therapy. After the end of
administration, arsenic rapidly falls back to normal levels. These
results are in agreement with the pharmacokinetics of rapid arsenic
absorption and of rapid arsenic elimination and demonstrate that
hair can reect very precisely absorbed arsenic variations. Of course
we have to insist on the fact that this is only true in the case of our
patients for whom external arsenic contamination was absent. It
would be probably more challenging to draw similar conclusions if
an external arsenic exposure had to be considered.
In Fig. 4 is shown a cartography of arsenic content in a hair
section cut in the middle of a hair of patient 1, in the part where
maximum arsenic concentration has been found by the longitudinal analysis. Arsenic is concentrated at the exterior part of the
cortex, just under the cuticle. Similar patterns are obtained with

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Fig. 3. Variation of as content in hair as a function of time. Patient 1 (left) received a continuous treatment, while patient 2 (right) a three cycles treatment.

RR
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Fig. 4. Optical microscopy view (left) and arsenic uorescence intensity (right) on a hair cut of patient 1. Arsenic is located at the periphery of the cortex.

Please cite this article in press as: I. Nicolis et al., Arsenite medicinal use, metabolism, pharmacokinetics and monitoring in human hair, Biochimie (2009), doi:10.1016/j.biochi.2009.06.003

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6. Conclusion

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Arsenic plays a dual role as environmental carcinogenic

pollutant and as a successful anticancer drug against APL (and
under investigation for other cancers). Its metabolism proceeds via
complicated enzymatic pathways, acting both as detoxication and
producing more toxic intermediates. We applied SXRF spectroscopy in human hairs, using them to monitor the history of arsenic
exposure by a longitudinal analysis but also to map the transversal
hair arsenic distribution and to proceed to a speciation of arsenic
compounds included in hair. Our kinetic results are in agreement
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speciation study agrees with previous observations of only inorganic arsenicals included in hair. Hair analysis by SXRF provides
data on all hair contained metals and in subsequent work we will
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elements. In conclusion the combination of the sensitivity and
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provides a reliable resource for trace element biomonitoring.

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rapid incorporation after injection. This result is also in agreement

with many published results, which state that inorganic trivalent
arsenic is the main form of arsenic in hair in contrast to urine which
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have been found in sheeps wool only when they were part of their
diet.[119] Both pentavalent and trivalent inorganic arsenic was the
major arsenical species in hair of people exposed by ingestion to
environmental arsenic of both valences.[79] We conrm that
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individual. We found a 1 eV shift between trivalent arsenic with
oxygen and trivalent arsenic with sulphur environments, in
agreement with literature values.[112] The hair XANES spectra
correspond to an environment of type oxygen or nitrogen and not
sulphur, suggesting arsenic is not linked to the keratin cysteine
residues. This is in agreement with the fact that cysteine residues in
hair keratine are engaged in cystine sulphur bridges and are not
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contained in the medulla has been found mainly bound to sulphur
by micro-XANES while bulk hair XANES indicated a larger
proportion of arsenate.[108] Once again, this difference could be
attributed to the different nature between human hair without
medulla and squirrels hair medulla.

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[6]
[7]

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Fig. 5. Micro-Xanes spectra at the arsenic Ka edge of high arsenic hair spot and of
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