Arsenic speciation in hair and nails of acute promyelocytic leukemia (APL) patients undergoing arsenic trioxide treatment

Author’s Accepted Manuscript Arsenic speciation in hair and nails of acute promyelocytic leukemia (APL) patients undergoing arsenic trioxide treatment Baowei Chen, Fenglin Cao, Xiufen Lu, Shengwen Shen, Jin Zhou, X. Chris Le www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(18)30259-5 https://doi.org/10.1016/j.talanta.2018.03.021 TAL18455

To appear in: Talanta Received date: 23 January 2018 Revised date: 8 March 2018 Accepted date: 8 March 2018 Cite this article as: Baowei Chen, Fenglin Cao, Xiufen Lu, Shengwen Shen, Jin Zhou and X. Chris Le, Arsenic speciation in hair and nails of acute promyelocytic leukemia (APL) patients undergoing arsenic trioxide treatment, Talanta, https://doi.org/10.1016/j.talanta.2018.03.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Arsenic speciation in hair and nails of acute promyelocytic leukemia (APL) patients undergoing arsenic trioxide treatment

Baowei Chen1, 2, Fenglin Cao3, Xiufen Lu2, Shengwen Shen2, Jin Zhou3,*, X. Chris Le2,*

1

Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Sun

Yat-Sen University, Guangzhou 510275, China 2

Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology,

University of Alberta, Edmonton, Alberta, T6G 2G3, Canada 3

Department of Hematology, Harbin Medical University, Affiliated Hospital 1, Harbin 150001,

Heilongjiang, China

*

Corresponding authors::

[email protected] [email protected]

1

ABSTRACT Arsenic in hair and nails has been used to assess chronic exposure of humans to environmental arsenic. However, it remains to be seen whether it is appropriate to evaluate acute exposure to sub-lethal doses of arsenic typically used in therapeutics. In this study, hair, fingernail and toenail samples were collected from nine acute promyelocytic leukemia (APL) patients who were administered daily with 10 mg of arsenic trioxide for up to 54 days. These hair and nail samples were analyzed for arsenic species using high performance liquid chromatography separation and inductively coupled plasma mass spectrometry detection (HPLC-ICPMS). Inorganic arsenite

was

Dimethylarsinic

the

predominant acid

form

(DMAV),

among

water-extractable

monomethylarsonic

acid

arsenicals. (MMAV),

monomethylarsonous acid (MMAIII), monomethylmonothioarsonic acid (MMMTAV), and dimethylmonothioarsinic acid (DMMTAV) were also detected in both hair and nail samples. This is the first report of the detection of MMAIII and MMMTAV as metabolites of arsenic in hair and nails of APL patients.

Keywords: Arsenic speciation; Hair; Nails; Acute promyelocytic leukemia; Biomarker

2

1. Introduction Acute promyelocytic leukemia (APL) is a distinctive type of acute myelocytic leukemia associated with reciprocal translocations between chromosomes 15 and 17. APL accounts for about 5-10% of all leukemia in adults [1-3]. Administration of all-trans retinoic acid (ATRA), as a first-line therapy, can induce 70% of APL patients to clinical remission [4]. However, the remaining 30% of APL patients are resistant to the treatment with ATRA. Arsenic trioxide

has been shown as an effective medicine

to overcome the resistance of APL to the ATRA treatment [5-9]. However, arsenic trioxide has been reported to cause serious side effects due to arsenic intoxication, e.g. gastrointestinal bleeding and disorders, liver enzyme elevation, renal failure, cardiac arrhythmia, and arsenic-induced neutropenia [10-13]. The same sub-lethal doses that were administered to all patients also resulted in sudden fatalities in several clinical cases [10, 14, 15]. Therefore, it is necessary to identify and quantify arsenic species in APL patients undergoing arsenic treatment in order to improve our understanding of arsenic metabolism and excretion in APL patients and facilitate timely clinical intervention and personalized therapy. Methylated arsenic metabolites have been detected in the urine and blood of APL patients [16-20]. However, arsenic speciation in the hair and nails of APL patients , particularly its temporal profile associated with arsenic treatment, remains to be studied. Arsenic in hair and nail clippings has been used as biomarkers for assessing human exposure to environmental arsenic [21-29]. Hair and nails are rich in cysteine-containing keratin. High affinity of arsenicals to cysteine facilitates arsenic accumulation in hair and nails [30, 31]. Arsenic concentration in the hair and nails reportedly correlated well with its concentration in other biological samples (e.g. urine, saliva, and blood) [21-23, 25, 26, 32], as well as exposure dose [31, 33]. Compared to other biological samples, hair and nails are less demanding in the collection, logistics and storage. Previous studies of arsenic speciation in hair and nails mainly dealt with chronic exposure to the low levels of arsenic in the environment [31]. In this study, APL patients were administered with a sub-lethal dose of arsenic for a shorter duration. The objective of this study was to determine 3

arsenic species in the hair, fingernails, and toenails of APL patients who were undergoing arsenic treatment for up to 54 days. This information should contribute to the understanding of arsenic metabolism in patients treated with therapeutic doses of arsenic.

2. Experimental 2.1 Reagents MMAIII and dimethylarsinous acid (DMAIII) were synthesized in the iodide form of monomethyldiiodoarsine

(CH3AsI2)

and

dimethylmonoiodoarsine

((CH3)2AsI)

according to the literature [34] and kept at 4 °C and -20 °C, respectively. MMMTAV and DMMTAV standards were prepared according to a reported method [35, 36] and stored at -20 °C. Their stock solutions were freshly prepared in deionized water. Stock solutions of other standards (1000 As mg/L) were prepared by dissolving appropriate amounts of AsIII, AsV, and DMAV (Aldrich) and MMAV (Chem Service) in deionized water. Working solutions were freshly prepared by serial dilutions in deionized water. Tetrabutylammonium hydroxide (Aldrich), malonic acid (Aldrich), and HPLC grade methanol (Fisher) were used to prepare the HPLC mobile phase. The mobile phase was filtered through a 0.45 μm membrane. Standard reference materials SRM1640 (Trace Elements in Natural Water) and CRM No. 18 Human Urine (Japan Environment Agency) were used for analytical quality control. Our replicate analyses (n=6) of CRM No.18 urine showed the presence of DMAV at the concentration of 31 ± 1 μg/L, which is in good agreement with the certified value of 36 ± 9 μg/L in the form of DMAV.

2.2 Participating APL patients Nine APL patients, two males and seven females (Table 1), were undergoing arsenic treatment in the Harbin Medical University Hospital, China. Informed consent from each of patients was acquired prior to the study which was conducted in compliance with the guidelines and regulation of both the Ethical Review Boards of the University of Alberta and Harbin Medical University Hospital. Demographic 4

information of APL patients participating in this study is presented in Table 1. Seven patients were newly diagnosed with APL, and two patients (patient 6 and 7) returned for arsenic treatment after a relapse. In the previous treatment, Patient 6 had been administered with the daily 10 mg dose of arsenic for 13 days. There was no medical record available for patient 7 regarding his previous arsenic treatment history. A 10-mL aqueous solution containing 10 mg arsenic trioxide (7.5 mg arsenic) was used as the daily dose of arsenic treatment. This solution was added into 500 mL of 5% glucose normal saline solution for intravenous infusion. The infusion usually lasted 2 to 4 h. Patients 4, 5 and 9 were administered arsenic over a period of as long as 18 h because of the concern of serious side effects on these patients if arsenic was administered within the shorter time. White blood cell counts and routine blood analysis were carried out daily for monitoring the patients’ health.

2.3 Hair and nail sample collection Hair (n=14), fingernail (n=10), and toenail (n=10) samples were collected from all nine APL patients (two males and seven females) (Table 1) who were undergoing arsenic treatment in the Harbin Medical University Hospital, China. Hair samples were collected near the scalp, and nail clipping (toenails and fingernails) were initially collected from the fingers of the left hand or toes of the left foot. After 4 days of arsenic administration, hair and nail samples were collected again. Nail samples were collected from the right hand or foot this time. All samples were individually stored in sealed plastic bags at -20 °C. The weights of hair samples were in the range of 29.5 to 143.9 mg, and those of nail samples were from 4.8 to 61.3 mg.

2.4 Washing and pretreatment of hair and nails for arsenic speciation Hair and nail clippings were washed according to a protocol adopted by the International Atomic Energy Agency. This protocol includes washings with analytical grade acetone once, deionized water three times, and acetone once again. Hair and nail samples are required to be in thorough contact with washing solvents under constant shaking for 10 min at room temperature. After washing, hair and nail 5

samples were air dried at room temperature and cut into smaller pieces for subsequent extraction of arsenic. Hair (~30-100 mg) or nail (~5-60 mg) samples were initially cut into ~0.5 cm and ~0.2 cm piece in length, respectively, and then were placed into a 15-mL centrifuge tube. Either 3 mL or 1 mL deionized water was added to the tube containing the hair or nail sample. These centrifuge tubes were placed in a hot water bath at 90 °C according to a reported method [21], with an extraction time of 6 h for hair and 1 h for nails. The extracts from each sample were immediately analyzed for arsenic species using HPLC-ICPMS. The residues were digested with acids for subsequent analysis of total arsenic using ICPMS.

2.5 Determination of arsenic remained in the residues The residues from the above extractions were digested with a mixture of 1 mL concentrated nitric acid and 3 mL concentrated sulfuric acid at 150 °C for 12 h. The mixture of acids was heated to boil and the acids were evaporated until approximately 300 μL solution remained. The digestion solution was diluted to 5 mL with deionized water and analyzed for total arsenic using ICPMS.

2.6 Arsenic speciation analysis by HPLC-ICPMS Separation of arsenic species was performed using ion-pair chromatography on a reversed-phase column (Phenomenex, ODS-3, 150×4.6 mm, 3 μm). The mobile phase contained 5 mM tetrabutylammonium hydroxide, 5% methanol and 3 mM malonic acid (pH 5.65) and ran at a flow rate of 1.2 mL/min. The column temperature was maintained at 50 °C. The column was equilibrated with the mobile phase at least for 0.5 h before sample injection. An aliquot of 20 μL sample was injected. The effluent from analytical column was directly introduced, using a PEEK tubing, into the nebulizer of an Agilent 7500ce octopole reaction system ICPMS. ICPMS was operated with the helium mode. The use of helium (3.5 mL/min) in the octopole reaction cell was to reduce polyatomic (ArCl+) interference on the determination of As+. The ICP was operated at a radio frequency power of 1550 W and a flow rate of 6

argon carrier gas of 0.9-1.0 L/min. Arsenic was monitored at m/z 75. Chromatograms were recorded by ICPMS ChemStation (Agilent Technologies, Santa Clara, CA). Detection limits from HPLC-ICPMS analyses of individual arsenic species were 0.1 g/L of arsenic in the solution. 2.7 Statistical analysis The relationships of total arsenic, methylated arsenicals, and inorganic arsenic between different samples were analyzed using OriginPro 8.0 software (OriginLab Corporation). The linear correlation at the p <0.05 level (95% confidence interval) was considered statistically significant.

3. Results Fig. 1 shows typical chromatograms from HPLC-ICPMS analyses of hair, fingernails, and toenails of APL patients who were undergoing arsenic treatment. AsIII, MMAIII, DMAV, MMAV, an unknown arsenic species (U1, peak 5), DMMTAV, AsV, MMMTAV, and a second unknown arsenic species (U2, peak 9) were detected in the hair (Fig. 1A). With the exception of the first unknown (peak 5), the other eight arsenic species were also detected in the fingernails (Fig. 1B) and toenails (Fig. 1C). The identities of MMAIII, DMMTAV, and MMMTAV were tested by spiking their respective authentic standards into the hair and nail samples containing them. Chromatogram overlays between the original hair sample and the same sample spiked with MMAIII (Fig. 2A), DMMTAV (Fig. 2B), and MMMTAV (Fig. 2C) are presented in Fig. 2. All spiked standards were co-eluted with the suspected arsenicals, resulting in an increase in the signals of the corresponding arsenic species. Simultaneous increase in the signals of MMAIII and MMAV, as shown in Fig. 2C, was due to the presence of MMAIII and MMAV as the decomposition products of the MMMTAV standard. AsIII, AsV, DMAV, and MMAV were frequently detected in the hair, fingernails, and toenails of APL patients (results summarized in Table 2). DMMTAV was also frequently detected in the nail samples. The mean concentrations of water-extractable

7

arsenicals from hair were 900.9 ng/g (AsIII), 90.1 ng/g (AsV), 9.4 ng/g (MMAV), 47.6 ng/g (DMAV), 36.8 ng/g (MMAIII), 56.1 ng/g (DMMTAV), and 11.1 ng/g (MMMTAV). The mean concentrations of AsIII in the fingernails and toenails were two times higher than that in the hair. On the contrary, the mean concentrations of AsV, DMAV, and DMMTAV in the fingernails and toenails were much lower than those in the hair. The toenails contained the highest concentrations of MMAV, MMAIII, and MMMTAV. Methylated arsenicals only accounted for a small fraction of water-extractable arsenic from the hair, fingernails, and toenails, with inorganic arsenicals being the predominant form (Fig. 3): 86.4% in the hair, 93.7% in the fingernails, and 89.3% in the toenails. Arsenic in the residues of hair, fingernails, and toenails after water extraction was also quantified after the residues were digested with concentrated nitric and sulfuric acids. The total arsenic concentrations in the hair and nails were obtained by the sum of arsenic concentration in the residue and all arsenic species in the extract. The mean concentration of total arsenic in the hair was 2.17 μg/g, which was much lower than in the fingernails (10.1 μg/g) and toenails (9.79 μg/g). The average extraction efficiencies of arsenic by water extraction were 32.5% for the hair (range: 4.0-57.7%), 22.6% for the fingernails (range: 10.0-79.4%), and 24.9% for the toenails (range: 10.0-48.6%). Arsenic concentrations in the hair, fingernails, and toenails of the seven newly diagnosed APL patients were plotted against the duration of the current treatment, as shown in Fig. 4. The treatment duration, according to the medical records of APL patients, was counted from the initial date of arsenic treatment to the date of sample collection. The results demonstrated that both the sum of water-extractable arsenic and total arsenic in the hair and nails were closely associated with the arsenic treatment duration. The treatment durations of three new APL patients (patient 1, 5, and 8) were less than 11 days. Arsenic concentrations in their hair, fingernails and toenails were 0.17, 1.33 and 0.41 μg/g, respectively. Arsenic concentrations in the hair and nails of new patients increased with the increase of the treatment duration. The highest concentration of total arsenic was 17.0 μg/g in the hair of patient 2 (44 days), 8

35.0 and 31.6 μg/g in the fingernails and toenails of patient 3 (54 days). The relapsed patient 6 stopped receiving arsenic treatment 77 days preceding the date of sampling, and did not resume arsenic treatment. The cessation of the arsenic treatment led to declined concentrations of total arsenic and methylated arsenicals in her hair (total arsenic 0.15 μg/g and methylated arsenicals below the detection limit), fingernails (8.60 and 0.07 μg/g), and toenails (28.3 and 0.15 μg/g). The percentages of methylated arsenic in the hair, fingernails, and toenails of new APL patients were also plotted with the treatment duration (Fig. 5). The results demonstrate that the percentages of methylated arsenic in the hair, fingernails, and toenails varied among APL patients who had different treatment durations. The hair and nails of APL patients who received no or only a short-term arsenic infusion contained the high percentages of methylated arsenic. With the prolongation of the arsenic treatment, the percentages of methylated arsenic in the hair, fingernails, and toenails of APL patients decreased. Correlation analysis of the concentrations of inorganic, methylated and total arsenic was conducted between the fingernails and toenails. Fig. 6 demonstrates a significant correlation between the fingernails and toenails at the 99% confidence interval for both inorganic arsenic (p <0.01, R=0.92) and methylated arsenic (p <0.01, R=0.78), as well as for total arsenic (p <0.01, R=0.80).

4. Discussion Nine APL patients were daily intravenously infused with arsenite (10 mg of arsenic trioxide dissolved in 10 mL water to form arsenous acid [AsIII(OH)3]). Inorganic AsIII can be readily transformed in humans through a series of biological processes [37, 38]. Methylated arsenicals have previously been detected as the metabolites that are present in various biological fluids, such as urine and blood [16-20]. AsIII and its metabolites are able to accumulate in the thiol-rich hair and nails [30]. AsIII, AsV, MMAV, and DMAV were detected in human hair, and AsIII, AsV, MMAV, DMAIII, DMAV, DMMTAV, and dimethyldithioarsinic acid (DMDTAV) in human nails [21-25, 39], all in environmentally chronic exposure scenarios. In the present clinical study, 9

AsIII, AsV, MMAIII, MMAV, MMMTAV, DMAV, and DMMTAV were determined in the hair, fingernails and toenails of APL patients. In particular, trivalent methylated and pentavalent thiolated arsenicals are subject to oxidation or decomposition during the sample pretreatment and analysis, thereby making it difficult to determine the presence of these arsenic species in biological samples [16, 21, 39]. MMAIII and MMMTAV were detected for the first time as arsenic metabolites in human hair and nails. Previously reported incubations of hair extracts separately with different arsenicals showed that MMAIII, MMMTAV, and DMMTAV could not be produced during the extraction and heating processes [40]. Therefore, the results of these detectable minor species (MMAIII, MMMTAV, and DMMTAV) in the hair and nails were likely produced from metabolism of arsenic inside the body of APL patients and accumulated in the hair and nails instead of being generated during arsenic extraction. Given the extraction inefficiency, original concentrations of MMAIII, MMMTAV, and DMMTAV in the hair and nails were expected to be higher. MMAIII has been previously detected in human urine [16, 41-43]. The present study shows that MMAIII is also present in the hair and nails of APL patients. Thiolated arsenicals have been detected in the urine of humans who were exposed to inorganic arsenic in drinking water [39, 44, 45]. In our study, the detection of MMMTAV and DMMTAV in the hair and nails of APL patients provided further support for the formation of these thiolated species in the biotransformation process of arsenic. MMAIII is more cytotoxic and genotoxic than inorganic arsenic or its pentavalent counterpart [46-48], and is more potent than arsenic trioxide in inducing the apoptosis of leukemia and lymphoma cells [49]. The thiolated arsenicals have also been shown to be as cytotoxic as trivalent oxygen-containing analogs [44, 50-53]. Thus MMAIII, MMMTAV, and DMMTAV, which are the minor arsenic species detected in this study, probably played an important role in eliciting apoptosis of cancerous leukemia cells [54]. Arsenic levels in the hair and nails were correlated with the duration of arsenic treatment. In the present study, APL patients were daily administered with sub-lethal 10

dose of AsIII (10 mg arsenic trioxide or 7.5 mg As). The concentrations of arsenic species and total arsenic in the hair and nails of these APL patients increased with the duration of arsenic treatment. Previous work has also showed correlation of arsenic in hair and nails with chronic exposure to arsenic from drinking water [21-24, 26, 55, 56]. Previous studies of arsenic species in the urine and saliva samples of APL patients, collected after an infusion of 7.5 mg arsenic (10 mg arsenic trioxide), demonstrated arsenic metabolism in the 24-hour period [16, 57]. In the present study, arsenic species in the hair and nails reflect the repeated daily infusion of arsenic and the consequence of arsenic metabolism and disposition to the hair and hails. Inorganic arsenic accounted for most of the arsenic in the hair and nails of APL patients, which was consistent with previous studies [21, 22, 24]. Inorganic arsenic has been demonstrated to be the dominant arsenic species in the urine and plasma of APL patients undergoing arsenic trioxide treatment [16, 19, 20]. The percentages of methylated arsenic in the hair and nails decreased with the duration of the arsenic treatment. A similar phenomenon was also observed in the urine of APL patients [16]. High levels of AsIII infused into the patients’ blood probably inhibited the activity of methyltransferases that were responsible for methylation of inorganic arsenic to the methylated arsenicals [58-60]. Arsenic speciation analysis

contributes to a better

understanding of arsenic exposure, metabolism, and health effects.

5. Conclusion Inorganic AsIII administered to APL patients were partially transformed into various arsenicals, including DMAV, MMAV, MMMTAV, MMAIII, and DMMTAV, which were detected in both hair and nail samples. The concentrations of these arsenic species and total arsenic in the hair and nails of APL patients were highly related to the duration of arsenic treatment. As compared to arsenic species in urine, which reflect arsenic exposure in the recent 1-3 days, arsenic species in the hair and nails represent arsenic exposure for a longer term. Determination of arsenic species in hair and nails complements the urinary arsenic speciation analysis, contributing to the monitoring of therapeutic treatment of API with arsenic. 11

Acknowledgements We thank the patients and hospital staff for participation in this study. This work was supported by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, the Canada Research Chairs Program, Alberta Innovates, and Alberta Health.

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14

Table 1. Summary of information of APL patients Duration of current treatment (days)

Past days of last treatment (duration)

Patient

Gender

Age

New or relapsed

1

M

30

new

11

no

2

M

50

new

44

no

3

F

19

new

54

no

4

F

24

new

13

no

5

F

24

new

0

no

6

F

29

relapsed

0

90(13)

7

F

36

relapsed

4

No record

8

F

40

new

9

no

9

F

52

new

16

no

15

Table 2. Summary of arsenic species in the hair, fingernails and toenails of APL patients (concentration in ng/g) Samples

Species

Mean

Range

Detectable percentage (%)

AsIII

900.9

5.1-8059.5

100

90.1

1.9-895.0

100

9.4

ND-60.7

85.7

47.6

ND-350.4

85.7

36.8

ND-104.4

35.7

11.1

ND-20.7

21.4

56.1

ND-84.0

14.3

U1

16.3

ND-28.5

14.2

U2

4.4

ND-13.8

35.7

AsIII

2284.9

65.8-13106.3

100

25.0

2.5-99.2

100

6.5

ND-22.6

90

23.1

ND-89.4

90

35.6

ND-62.9

40

7.7

ND-8.9

30

16.2

ND-52.6

90

U2

14.1

ND-24.4

50

AsIII

2469.3

27.5-13788.5

100

59.1

1.8-295.5

100

18.1

ND-45.7

90

35.4

ND-174.5

100

90.9

ND-285.2

40

12.9

ND-12.9

10

17.4

ND-44.5

80

35.0

ND-75.9

50

V

As

MMA DMA Hair

V

V

MMAIII

(n=14) MMMTA DMMTA

V

V

V

As

MMA

V

V

Fingernail

DMA

(n=10)

MMAIII MMMTA DMMTA

V

V

V

As

MMA

V

V

Toenail

DMA

(n=10)

MMAIII MMMTA DMMTA U2

V

V

ND means not detectable (below the detection limit of 1.0 ng/g).

16

1

3

(A) Hair

7

5 2

0

1

4

2

3

9

6

8

4

5

6

Retention time (min) 1 (B) Fingernail

3

7 4

0

1

2

3

6 9

4

5

6

Retention time (min)

1 (C) Toenail

2 3

7 4

0

1

2

3

6

4

9 8

5

6

Retention time (min)

Fig. 1. Chromatograms showing arsenic species in the hair, fingernails, and toenails of APL patients undergoing arsenic treatment. Speciation separation was achieved on an ODS column (150 mm × 4.6 mm, 3 μm particle size, Phenomenex). The mobile phase consisted of 5 mM tetrabutylammonium, 3 mM malonic acid, and 5% methanol (pH 5.65) and was run at a flow rate of 1.0 mL/min at 50 °C. Arsenic was detected at m/z 75 using an Agilent 7500ce octopole reaction system ICPMS. Peak 1-9 represented AsIII, MMAIII, DMAV, MMAV, U1 (unknown species 1), DMMTAV, AsV, MMMTAV, and U2 (unknown species 2), respectively. The dash line chromatograms were from the analysis of hair samples containing U1 and U2.

17

2

A

B 6

C 8 0

2

4

6

Retention time (min) Fig. 2. Chromatograms from HPLC-ICPMS analyses of hair samples (solid line) and the same samples spiked with authentic arsenic standards (dash line). Peak identities and HPLC-ICPMS conditions were the same as those in Fig. 1. (A) Sample spiked with MMAIII, (B) Sample spiked with DMMTAV, (C) Sample spiked with MMMTAV.

18

Relative percentage (%)

100

Unknown DMA MMA As(V) As(III)

75 50 25 0

Hair

Fingernail

Toenail

--

Fig. 3. The relative percentages of AsIII, AsV, MMA, DMA, and unknown arsenic species in the extract of the hair, fingernails, and toenails samples from APL patients.

19

Sum of water extractable As Total As

Arsenic concentration (ng/g)

1.0x104

Patient 5 3

5.0x10

8

Hair

Patient 2

Patient 3

1 4 9

0.0 6.0x104

Fingernail

4.0x104 2.0x104 0.0 4.0x104

Toenail 2.0x104 0.0

0

10

20

30

40

50

60

Treatment duration of time (Day)

Fig. 4. Arsenic concentrations in the hair, fingernails, and toenails of APL patients with increasing duration of arsenic treatment.

20

Relative percentage of methylated arsenicals (%)

40

30

Hair Fingernail Toenail

Patient 5 8 1 4

20

Patient 2

9

Patient 3

10

0 0

10

20

30

40

50

60

Treatment duration of time (day) Fig. 5. Percentages of methylated arsenicals in the hair, fingernails, and toenails of APL patients with increasing duration of arsenic treatment.

15

Arsenic in toenail (µg/g)

(A) Inorganic arsenic R =0.92 p <0.01

10

5

0 0

5

10

Arsenic in fingernail (µg/g)

21

15

Arsenic in toenail (µg/g)

0.6

(B) Methylated arsenic R =0.78 p <0.01

0.4

0.2

0.0 0.0

0.1

0.2

Arsenic in toenail (µg/g)

Arsenic in fingernail (µg/g)

(C) Total arsenic

40

R =0.80 p <0.01

30 20 10 0 0

10

20

30

40

Arsenic in fingernail (µg/g)

Fig. 6. Correlations of inorganic arsenic, methylated arsenic, and total arsenic concentrations between fingernails and toenails. Highlights:

Speciation analyses of hair and nail from acute promyelocytic leukemia (APL) patients reveal methylated and thiolated arsenic metabolites Concentrations of arsenicals in nine APL patients’ hair and nail increase with the duration of arsenic treatment Monomethylarsonous acid (MMAIII), monomethylmonothioarsonic

22

acid (MMMTAV), and dimethylmonothioarsinic acid (DMMTAV) are detected in the water-extracts of the patients’ hair and nail samples

23