- Email: [email protected]
Determination of Total Arsenic and Arsenic Metabolites in Human Liver Hepatocytes
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 37, Issue 1, January 2009 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2009, 37(1), 19–24.
RESEARCH PAPER
Determination of Total Arsenic and Arsenic Metabolites in Human Liver Hepatocytes CAO Xuan1,2, YU Jing-Jing2, WANG Geng3, YU Zhen-Hua2,4, YANG Huang-Hao2, XU Yuan-Yuan5, SUN Gui-Fan5, WANG Xiao-Ru1,2,* 1
College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266003, China First Institute Oceanography of State Oceanic Administration, Qingdao 266061, China 3 College of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China 4 Institute of Oceanology Chinese Academy of Science, Qingdao 266071, China 5 College of Pubic Health, China Medical University, Shenyang 110001, China 2
Abstract:
In this article, the effects of three digestion methods on the determination of arsenic in Chang liver hepatocytes after
ultrasonication were compared with direct sampling. The results showed that the efficiency of microwave digestion and obturator digestion was better than that of cold digestion and direct sampling. The daily precision (present as RSD) of As determination with the sample treatment of microwave digestion and obturator digestion was 2.14% and 1.21%, the interday precision of the determinations was 1.15% and 1.97%. The spike recovery for the total As in the sample is in the range of 95.7%–108.1%. The As detection limits with these four sample treatment methods (including direct sampling) were among 0.74–0.93 ȝg l–1. In addition, arsenic species in Chang liver hepatocytes were also analyzed using the hyphenated technique of high-performance liquid chromatography coupled with inductively coupled plasma-mass spectrometry. The experimental results indicated that dimethylarsinic acid and an intermediate metabolite of DMA were found in Chang liver hepatocytes besides inorganic arsenic (As(III) and As(V)). Key Words:
Cell digestion; High performance liquid chromatography; Inductively coupled plasma-Mass spectrosmeter; Chang
liver hepatocytes; Arsenic speciation
1
Introduction
Arsenic is one of the most abundant elements in human body, and also a kind of carcinogenic substance[1,2]. Long-term arsenic exposure would be resulting in human health risks, especially tissue and organ damages[3]. Nowadays, with the concern of human health, more and more researches were devoted to uncover the process of arsenic metabolism in human. However, it is a tough work. As the toxicity of arsenic is extreme, it was immoral to study arsenic metabolism in human body. Furthermore, arsenic metabolism was varied with its species dramatically. Some literatures indicated that arsenic metabolism in chimps[4], which were most closed to human, were very different from human. Thereby, animal experiments
could only approximately reflect the arsenic transformations in human body, but not details. Recently, scientists in this field inclined to choose human cells, which derived from human as the target to investigate arsenic metabolism in human. Compared to the animal experiments, human cells were derived from human, which should be right to reflect its metabolism pathway in human body. Arsenic metabolism study was based on the authentic determination, which included total arsenic (AsT) determination and arsenic speciation analysis. Unfortunately, so far, there seemed no effective AsT determination methods available because of the low arsenic concentration and high matrix in the cells. For AsT determination, the cells should be pretreated into a homogeneous and steady solution, which is the crucial step of
Received 6 May 2008; accepted 22 September 2008 * Corresponding author. Email: [email protected] This work was supported by the National Natural Science Foundation of China (No. 20675021). Copyright © 2009, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(08)60079-1
CAO Xuan et al. / Chinese Journal of Analytical Chemistry, 2009, 37(1): 19–24
AsT determination. The common methods were ultrasonication or cold digestion[5]. These pretreatments were simple, but the efficiency was low. In this study, three cell homogeneity methods were compared based on cells ultrasonication to evaluate the homogeneity efficiency and their effects on the AsT determination. An authentic AsT method was established to analyze arsenic in Chang liver hepatocytes. Based on AsT, a qualitative analysis of arsenic species was also carried out to initiate the arsenic metabolites in Chang liver hepatocytes.
2 2.1
Experimental
and 20 ȝM) for 24 h at 37 °C. The culture media was then removed, and the hepatocytes were washed with phosphate-buffered saline (PBS) three times and then were harvested using cell brush. Approximately 2.5 × 106 hepatocytes in each well were dissolved into 1 ml of PBS separately and were subjected to ultrasonication. The hepatocytes after ultrasonication were filtered through 0.45 ȝm membrane, and the membrane was washed with PBS for three times. Finally, clear heptatocyte solution of 2 ml was formed, which was divided into equally two portions and subjected to the determination of AsT and arsenic speciation. These samples were stored in –70 °C before analysis.
Instruments and reagents 2.3
An Agilent 1100 series high-performance liquid chromatography (HPLC) system consisting of a binary pump, a vacuum degasser, and a manual-sampler with a 20 ȝl injection loop was used in HPLC analysis. A PRP-X100 anion exchange column (Hamilton, Reno, NV, USA) was used for the separation of arsenic species. Arsenic determination was performed by an Agilent 7500a ICP-MS (Agilent Technologies, USA) equipped with a Babington nebulizer, a glass double-path spray chamber and a standard quartz torch. Microwave digestion of cells was performed on Speed wave MW-3+ Microwave System (Berghof, Germany). Degassed, ultrapure 18 Mȍ water (DDI; Millipore, Bedford, MA) was used throughout. The stock solutions of 1000 mg l–1 arsenic were prepared using the following standard compounds. Arsenite trioxide As(III) and arsenate oxide As(V) were purchased from Alfa Aesar (Ward Hill, MA). Dimethylarsinic acid (DMA) was purchased from Acros Organics (NJ, USA). Monomethylarsinic acid (MMA) was prepared by Tsinghua University (Beijing, China). All the stock solutions were stored in a dark room at 4 °C, and the standard solutions containing arsenic species were prepared fresh before use. RPMI 1640 medium was purchased from Gibco (Grand Island, USA). Strong oxidants included 65% nitric acid and 30% hydrogen peroxide used for cell digestion were purchased from Merck (Darmstadt, Germany). Chang liver hepatocytes were purchased from Shanghai Cell Bank, Chinese Academy of Medical Sciences. 2.2
Cell preparation and incubation
Human Chang liver cell lines were cultured in RPMI 1640 medium. A total of 1 × 105 viable cells were seeded per well in 6-well plates. The culture media were supplemented with 10% fetal calf serum, penicillin (100 units ml–1), streptomycin (100 ȝg ml–1, and 0.03% glutamine. The cultures were maintained at 37 °C in a water-saturated atmosphere containing 5% CO2 and 95% atmosphere. After cell attachment⩖the hepatocytes were incubated with various arsenite initial concentrations (1, 5, 10
Determination of total arsenic
The AsT in the Chang liver cells were determined with ICP-MS after digestion using four different methods: direct sampling introduction, cold digestion, obturator digestion, and microwave digestion. 2.3.1
Direct injection (DJ)
One milliliter of hepatocyte solution, approximately 2.5 × 106 hepatocytes, was shaken up and diluted 10 fold for direct analysis by ICP-MS. 2.3.2
Cold digestion (CD)
One milliliter of hepatocytes solution, 1 ml of concentrated HNO3, and 200 ȝl of H2O2 were added into a PET centrifuge tube. The tube were sealed off and stood for 24 h. Shaking the tube to disperse the sediment timely during this period. This solution was diluted 10 fold with deionized water and then determined using ICP-MS when the sediment could not be observed. 2.3.3
Obturator digestion (OD)
One milliliter of hepatocyte solution and 2.0 ml concentrated HNO3 were added into a PTFE bomb. After 10 min, the bombs were sealed off and put into an oven at 80 °C for 2 h. Afterwards, the bombs were cooled down and then placed in the oven at 170 °C for another 2 h. After cooling down the bomb, 200 ȝl H2O2 was added onto the residue inside the bomb to form a clear solution. This solution was diluted to 10 fold for ICP-MS analysis. 2.3.4
Microwave digestion (MD)
Microwave digestion was performed using Speed wave MW-3+ microwave digestion system (Berghof, Germany). One milliliter of hepatocyte solution and 2 ml of concentrated HNO3 were added into a PTFE bomb. The digestion temperature increased as the gradient of 100, –180, –200, and
CAO Xuan et al. / Chinese Journal of Analytical Chemistry, 2009, 37(1): 19–24
–210 °C, every temperature stood for 10 min. After digestion, 200 ȝl of H2O2 was added and allowed to stand for 5 min to enhance the digestion efficiency and to obtain a clear solution. This digestion solution was diluted 10 fold before the analysis. 2.4
Analysis of arsenic speciation
2.4.1
Sample pretreatment
Table 1 Instrumental parameters for total As determination and As speciation analysis Total arsenic ICP-MS RF power (W) RF matching (V) Carrier gas flow rate (l min–1) Peristaltic pump flow rate Monitored signals
Agilent 7500a 1350 1.6 1.14 0.1 rps 75 As, 72Ge, 35Cl, 77Se, 83Kr and 34S
Arsenic speciation
The samples were thawed at 4 °C in 99.99% nitrogen atmosphere, which could prevent the oxidation of arsenic metabolites, and then samples were filtered using 0.45 ȝm membrane for the final analysis. 2.4.2
Chromatography and ICP-MS operating conditions
An anion exchange column (Hamilton PRP-X100) was applied to separate arsenic metabolites in hepatocytes. Both chromatography and ICP-MS operating conditions are listed in Table 1.
3 3.1
Results and discussion Comparison of four AsT methods
To obtain the accurate total element concentration in biological samples, pretreatment was a crucial step. Generally, digestion is the best choice. Digestion includes wet digestion and dry digestion. For AsT, dry digestion could be more efficient, and the operation procedure is simple. However, As could be sublimed or easier to form arsenic dimer and arsenic oxide at very low temperature. These substances escaped and caused arsenic loss in the procedure of dry digestion. Therefore, dry digestion was not suitable for arsenic digestion. The recommended arsenic digestion method was wet digestion. Wet digestion method was performed in an enclosed space. By strong acid and oxidants, the samples were digested to form homogeneous and steady solution. Wet digestion could be classified to obturator digestion, microwave digestion, and cold digestion, etc. For obturator and microwave digestions, the samples were both digested using strong acid in an enclosed space at high temperature. However, there was difference in the heating modes. The former was external heating, that is, the samples in obturator were heated in ovens or muffles to achieve digestion. The latter was internal heating that used the penetrability and activation of microwave to achieve digestion.
HPLC Analytical column Flow rate(ml min–1) Injection volume (μl) Mobile phase A Mobile phase B Gradient program
Agilent 1100 Series Hamilton PRP-X100 (250 mm × 4.1 mm, 10 μm) 1.8 25 H2O 50 mM (NH4)2CO3˄pH=7.4 adjusted by acetic acid˅ Time (min)
A (%)
B (%)
0 15 30
100 0 100
0 100 0
While cold digestion was suitable for the samples, which contain relatively less organics. In cold digestion, the samples were digested without heating but just by the strong acid or oxidants. In this study, these digestion methods were investigated systematically to find out the optimal digestion methods for the hepatocytes. Table 2 shows the comparison results of AsT determination with the four digestion methods. Among these methods, obturator digestion and microwave digestion methods showed the excellent performance than any others. The direct injection and cold digestion methods were inferior. Especially for direct injection, approximately only 50% AsT was obtained compared with microwave digestion. The RSDs of AsT measurement with obturator digestion and microwave digestion methods were lower than that of the others, whereas the highest was obtained when using direct injection. This suggested that the cells were not broken up thoroughly or samples were not well homogenized after ultrasonication, which may result in the poor accuracy and stability of AsT determination with direct injection. The RSD% of AsT determination with cold digestion method was modest; cold digestion could enhance the homogeneity of the samples, however, compared with heating digestion (obturator and microwave digestion), and the accuracy and stability of the determination with cold digestion could not meet the need of analytical chemistry. There was no obvious difference in the results from Table 2.
Table 2 Comparison of 4 total arsenic determination methods (ȝg l–1) Cultured conca Blank 1 ȝM group 5 ȝM group 10 ȝM group 20 ȝM group a
DJ 2.01 ± 1.99 12.2 ± 7.2 24.4 ± 9.3 50.0 ± 12 98.2 ± 9.9
CD 2.31 ± 1.60 14.5 ± 5.3 33.1 ± 4.1 78.8 ± 8.2 110.3 ± 8.1
MD 2.64 ± 0.34 18.1 ± 0.7 48.2 ± 1.3 99.8 ± 2.7 154.0 ± 2.1
OD 2.75 ± 0.49 17.7 ± 1.0 48.8 ± 1.2 101.8 ± 3.8 156.2 ± 3.2
mean ± standard deviation, n = 3; DJ: represents direct injection; CD: represents cold digestion; MD: represents microwave digestion; OD: represents obturator digestion.
CAO Xuan et al. / Chinese Journal of Analytical Chemistry, 2009, 37(1): 19–24
With both the methods of obturator digestion and microwave digestion nevertheless, obturator digestion consume relatively long time, which might increase the possibility of pollution. In this study, the microwave digestion was chosen to treat the sample prior to the determination of AsT in Chang liver hepatocytes. 3.2
Interferences elimination
The interference was the main obstacle to interfere the accuracy of arsenic determination. There are two types of interferences in ICP-MS, mass spectroscopic and nonmass spectroscopic interferences. Mass spectroscopic interference of arsenic was derived from 35Cl, which was abundant in biological samples. The isotope of 35Cl could combine with carrier gas (40Ar) in plasma to form 40Ar35Cl, and a quadrupole mass filter could not distinguish 75As and 40Ar35Cl. To solve this problem, the samples were diluted to decrease the concentration of 35Cl, and meanwhile, ICP-MS operating conditions were optimized and interference equation 75As = 75 M – 77M (3.127) + 82M (2.733) – 83M (2.757) was used to resolve this polyatomic interference. Nonmass spectroscopic interferences was mainly derived from sample matrix, and high matrix of biological samples would suppress the signals of arsenic. In this study, the isotope of 89Y was used as an internal standard to adjust this suppression. 3.3
Linear range and method precision
A series of 0–200 ȝg l–1 arsenic standard solutions was prepared for the calibration curve. The excellent linear correlation coefficient was obtained (0.9998). Dual mode detector and nine order of dynamic range of ICP-MS confirmed arsenic determination validation. The measurement precision was evaluated by determination of 1 ȝM arsenic group cells using four different methods of sample pretreatment for seven times. The RSDs of the measurement with each method were listed in Table 3.
Compared with direct injection and cold digestion, the RSDs of obturator and microwave digestion methods were superior, which were 1.21% and 2.14%, respectively. Besides, long-term detection precision was also investigated in six days (one time determination each day), and the results of interday precision of six days are also listed in Table 3. The best long term detection precision could be obtained with Microwave digestion method, while the worst precision was observed with using direct injection (RSD = 12.6%). Compared with obturator method, microwave digestion shows the preferable performance in long-term determination, although its day precision was slightly inferior to obturator digestion. 3.4
Detection limits and spike recovery
The detection limits were confirmed by 3 × SD (Standard Deviation) of eleven times independent determination of blank sample, and the results are shown in Table 4. The accuracy of the method for the As determination was assessed by the spike recovery because of the lack of cell-certificated relative materials (CRMs). Approximately 100% background arsenic concentration was spiked into 5 ȝM arsenic group cells. After those of four pretreatment methods, recovered spike concentrations were determined to calculate the recovery ratio, which are shown in Table 4. The spike recoveries ranged from 84.4%–108.1%, which indicate that the method accuracy is quite satisfactory except using the direct injection method (64.0%). 3.5
Arsenic speciation in Chang liver hepatocytes
Inorganic arsenic could be metabolized to yield monomethylated, dimethylated metabolites, and possibly some intermediate arsenic motablites. This process was termed as biomethylation, which is a major metabolic pathway for inorganic arsenic in humans and in most animal species[6–8], although the detailed process was not well understood yet.
Table 3 Measurement precision and inter-day precision Method DJ CD MD OD
Precision (RSD, %)
Detection limit (ȝg l–1)
1st day (ȝg l–1)
2nd day (ȝg l–1)
3rd day (ȝg l–1)
4th day (ȝg l–1)
5th day (ȝg l–1)
6th day (ȝg l–1)
Inter-day precision (RSD, %)
7.80 5.60 2.14 1.21
0.93 0.74 0.91 0.79
12.3 14.6 17.9 17.6
9.40 13.3 18.3 17.5
11.5 15.6 18.0 17.8
10.5 15.8 18.4 18.0
8.70 12.0 18.2 17.0
10.4 13.1 18.4 17.4
12.6 10.9 1.15 1.97
DJ: represents direct injection; CD: represents cold digestion; MD: represents microwave digestion; OD: represents obturator digestion.
Table 4 Detection limits and spike recovery Method DJ CD MD OD
Detection limits (ȝg l–1) 0.93 0.74 0.91 0.79
Spike recovery Original content (ȝg l–1) 24.4 35.7 48.9 49.2
Added (ȝg l–1) 20 30 40 40
Found (ȝg l–1) 28.4 55.3 96.1 88.4
Recovery (%) 64.0 84.1 108.1 95.7
CAO Xuan et al. / Chinese Journal of Analytical Chemistry, 2009, 37(1): 19–24
Biomethylation mainly occurred in liver[9,10], and the determination and distinguishing of those biomethylation metabolites would reflect the degree of arsenic metabolism in the organism. Furthermore, the qualitative characterization of methylated arsenic metabolites in liver might give intuitive support to uncover biomethylation mechanism. In this study, an anion exchange chromatography was used for the separation of six arsenic species, such as As(III), As(V), DMA, MMA, AsB, and AsC. Deionized water (A) and 50 mM (NH4)2CO3 (B) were used as the mobile phases. The grade elution conditions are listed in Table 1, and Fig.1 showed the HPLC-ICP-MS spectrum of those six arsenic species. All these arsenic species referred in this study could be baseline separation using this chromatography conditions. Using these chromatography conditions, arsenic species in 1.0 ȝM arsenic group Chang liver hepatocytes were analyzed. To prevent the interference of 40Ar and 35Cl[10], the signals of 75 As, 72Ge, 35Cl, 77Se and 83Kr were monitored simultaneously. The arsenic species in Chang liver hepatocytes are illustrated in Fig.2. Among all the species, As(V) was found to be the main component in hepatocytes. The observed As(V) possibly be derived from the oxidation of As(III), which was not metabolized by hepatocytes. Besides As(V), As(III), DMA, and an unknown arsenic peak were also found in hepatocytes. As we know, the arsenic metabolism in human liver was not well understood so far. But arsenic biomethylation did exist in liver when inorganic arsenics were ingested into human. As(V) was first transformed to As(III) and then methylated to MMA; MMA, which was a mark of first step methylation, could be methylated again to DMA through a series of intermediate arsenic metabolites. These two steps of methylation had been confirmed elsewhere[11,12]. As shown in Fig.2, the finding of DMA showed that the methylation did occur in Chang liver hepatocytes. Interestingly, instead of MMA, an unknown arsenic peak was also found in the chromatogram. It deduced that this unknown peak might be intermediate metabolites of MMA because MMA was not observed in the chromatogram. To prove this hypothesis, an appropriate content of H2O2 was added into the samples and then made stable for 4 h. Arsenic species in hepatocytes after H2O2 treatment are shown in Fig.3. Compared to Fig.2, both As(III) and the unknown peak were disappeared. As a counterpart, the peak area of As(V) increased about 25%, which is resulted from the oxidation of As(III). Meanwhile, MMA was observed. There are some literatures[13], indicating that H2O2 could break the bond between the arsenic and sulfhydryl group. Therefore, this phenomenon testified that the unknown peak was the intermediate metabolites of MMA. This unknown arsenic species possessed significant importance owing to the identification of arsenic intermediate metabolites that were the key to explain arsenic metabolism in the human body[11,12]. If this arsenic species could be identified exactly, it might give a strong support to well understand the arsenic metabolism in human body.
Fig.1 HPLC-ICP-MS spectrum of 6 mixed As species standards 1. AsC; 2. AsB; 3. As(III); 4. DMA; 5. MMA; 6. As(V)
Fig.2
HPLC-ICP-MS chromatographic spectrum of As species compounds in 1.0 ȝM group
Fig.3
HPLC-ICP-MS chromatographic spectrum of As species compounds in 1.0 ȝM group after H2O2 treatment
4
Conclusion
The effects of three digestion methods (cold digestion, obturator digestion, and microwave digestion) on the ICP-MS determination of arsenic in Chang liver hepatocytes after ultrasonic disintegration were systematically studied. The comparison results indicate that the better performance on the arsenic determination in Chang liver hepatocytes, including digestion efficiency, detection precision, interday precision, and spike recovery, could be expected with obturator and microwave digestions. However, when digestion time of samples was taken into account, microwave digestion was
CAO Xuan et al. / Chinese Journal of Analytical Chemistry, 2009, 37(1): 19–24
finally recommended as the best method to determine the arsenic in Chang liver hepatocytes. Furthermore, a HPLC-ICP/MS hyphenated technique was established and then used to analyze the arsenic species in Chang liver cells. An unknown arsenic intermediate metabolite was found, which might supply some useful information to well understand arsenic metabolism in human body.
Chinese Medical Equipment, 2004, 1(4): 39–43 [6]
Aposhian H V. Annual Review of Pharmacology and Toxicology, 1997, 37(1): 397–419
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Radabaugh T R, Sampayo-Reyes A, Zakharyan, R A, Aposhian H V. Chemical Research in Toxicology, 2002, 15(5): 692–698
[8]
Thomas D J, Styblo, M, Lin S. Toxicology and Applied Pharmacology, 2001, 176(2): 127–144.
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Suzuki K T, Katagiri A, Sakuma Y, Ogra Y, Ohmichi M.
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Aposhian H V, Gurzau E S, Le X C, Gurzau A, Healy S M, Lu X, Ma M, Yi P L, Zaleharyan R A, Maiorino R M, Tircus
198(3): 268–271 [4]
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Water. Washington, DC: National Academy Press, 1999
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Cite this article as: Chin J Anal Chem, 2009, 37(1), 19–24.
RESEARCH PAPER
Determination of Total Arsenic and Arsenic Metabolites in Human Liver Hepatocytes CAO Xuan1,2, YU Jing-Jing2, WANG Geng3, YU Zhen-Hua2,4, YANG Huang-Hao2, XU Yuan-Yuan5, SUN Gui-Fan5, WANG Xiao-Ru1,2,* 1
College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266003, China First Institute Oceanography of State Oceanic Administration, Qingdao 266061, China 3 College of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China 4 Institute of Oceanology Chinese Academy of Science, Qingdao 266071, China 5 College of Pubic Health, China Medical University, Shenyang 110001, China 2
Abstract:
In this article, the effects of three digestion methods on the determination of arsenic in Chang liver hepatocytes after
ultrasonication were compared with direct sampling. The results showed that the efficiency of microwave digestion and obturator digestion was better than that of cold digestion and direct sampling. The daily precision (present as RSD) of As determination with the sample treatment of microwave digestion and obturator digestion was 2.14% and 1.21%, the interday precision of the determinations was 1.15% and 1.97%. The spike recovery for the total As in the sample is in the range of 95.7%–108.1%. The As detection limits with these four sample treatment methods (including direct sampling) were among 0.74–0.93 ȝg l–1. In addition, arsenic species in Chang liver hepatocytes were also analyzed using the hyphenated technique of high-performance liquid chromatography coupled with inductively coupled plasma-mass spectrometry. The experimental results indicated that dimethylarsinic acid and an intermediate metabolite of DMA were found in Chang liver hepatocytes besides inorganic arsenic (As(III) and As(V)). Key Words:
Cell digestion; High performance liquid chromatography; Inductively coupled plasma-Mass spectrosmeter; Chang
liver hepatocytes; Arsenic speciation
1
Introduction
Arsenic is one of the most abundant elements in human body, and also a kind of carcinogenic substance[1,2]. Long-term arsenic exposure would be resulting in human health risks, especially tissue and organ damages[3]. Nowadays, with the concern of human health, more and more researches were devoted to uncover the process of arsenic metabolism in human. However, it is a tough work. As the toxicity of arsenic is extreme, it was immoral to study arsenic metabolism in human body. Furthermore, arsenic metabolism was varied with its species dramatically. Some literatures indicated that arsenic metabolism in chimps[4], which were most closed to human, were very different from human. Thereby, animal experiments
could only approximately reflect the arsenic transformations in human body, but not details. Recently, scientists in this field inclined to choose human cells, which derived from human as the target to investigate arsenic metabolism in human. Compared to the animal experiments, human cells were derived from human, which should be right to reflect its metabolism pathway in human body. Arsenic metabolism study was based on the authentic determination, which included total arsenic (AsT) determination and arsenic speciation analysis. Unfortunately, so far, there seemed no effective AsT determination methods available because of the low arsenic concentration and high matrix in the cells. For AsT determination, the cells should be pretreated into a homogeneous and steady solution, which is the crucial step of
Received 6 May 2008; accepted 22 September 2008 * Corresponding author. Email: [email protected] This work was supported by the National Natural Science Foundation of China (No. 20675021). Copyright © 2009, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(08)60079-1
CAO Xuan et al. / Chinese Journal of Analytical Chemistry, 2009, 37(1): 19–24
AsT determination. The common methods were ultrasonication or cold digestion[5]. These pretreatments were simple, but the efficiency was low. In this study, three cell homogeneity methods were compared based on cells ultrasonication to evaluate the homogeneity efficiency and their effects on the AsT determination. An authentic AsT method was established to analyze arsenic in Chang liver hepatocytes. Based on AsT, a qualitative analysis of arsenic species was also carried out to initiate the arsenic metabolites in Chang liver hepatocytes.
2 2.1
Experimental
and 20 ȝM) for 24 h at 37 °C. The culture media was then removed, and the hepatocytes were washed with phosphate-buffered saline (PBS) three times and then were harvested using cell brush. Approximately 2.5 × 106 hepatocytes in each well were dissolved into 1 ml of PBS separately and were subjected to ultrasonication. The hepatocytes after ultrasonication were filtered through 0.45 ȝm membrane, and the membrane was washed with PBS for three times. Finally, clear heptatocyte solution of 2 ml was formed, which was divided into equally two portions and subjected to the determination of AsT and arsenic speciation. These samples were stored in –70 °C before analysis.
Instruments and reagents 2.3
An Agilent 1100 series high-performance liquid chromatography (HPLC) system consisting of a binary pump, a vacuum degasser, and a manual-sampler with a 20 ȝl injection loop was used in HPLC analysis. A PRP-X100 anion exchange column (Hamilton, Reno, NV, USA) was used for the separation of arsenic species. Arsenic determination was performed by an Agilent 7500a ICP-MS (Agilent Technologies, USA) equipped with a Babington nebulizer, a glass double-path spray chamber and a standard quartz torch. Microwave digestion of cells was performed on Speed wave MW-3+ Microwave System (Berghof, Germany). Degassed, ultrapure 18 Mȍ water (DDI; Millipore, Bedford, MA) was used throughout. The stock solutions of 1000 mg l–1 arsenic were prepared using the following standard compounds. Arsenite trioxide As(III) and arsenate oxide As(V) were purchased from Alfa Aesar (Ward Hill, MA). Dimethylarsinic acid (DMA) was purchased from Acros Organics (NJ, USA). Monomethylarsinic acid (MMA) was prepared by Tsinghua University (Beijing, China). All the stock solutions were stored in a dark room at 4 °C, and the standard solutions containing arsenic species were prepared fresh before use. RPMI 1640 medium was purchased from Gibco (Grand Island, USA). Strong oxidants included 65% nitric acid and 30% hydrogen peroxide used for cell digestion were purchased from Merck (Darmstadt, Germany). Chang liver hepatocytes were purchased from Shanghai Cell Bank, Chinese Academy of Medical Sciences. 2.2
Cell preparation and incubation
Human Chang liver cell lines were cultured in RPMI 1640 medium. A total of 1 × 105 viable cells were seeded per well in 6-well plates. The culture media were supplemented with 10% fetal calf serum, penicillin (100 units ml–1), streptomycin (100 ȝg ml–1, and 0.03% glutamine. The cultures were maintained at 37 °C in a water-saturated atmosphere containing 5% CO2 and 95% atmosphere. After cell attachment⩖the hepatocytes were incubated with various arsenite initial concentrations (1, 5, 10
Determination of total arsenic
The AsT in the Chang liver cells were determined with ICP-MS after digestion using four different methods: direct sampling introduction, cold digestion, obturator digestion, and microwave digestion. 2.3.1
Direct injection (DJ)
One milliliter of hepatocyte solution, approximately 2.5 × 106 hepatocytes, was shaken up and diluted 10 fold for direct analysis by ICP-MS. 2.3.2
Cold digestion (CD)
One milliliter of hepatocytes solution, 1 ml of concentrated HNO3, and 200 ȝl of H2O2 were added into a PET centrifuge tube. The tube were sealed off and stood for 24 h. Shaking the tube to disperse the sediment timely during this period. This solution was diluted 10 fold with deionized water and then determined using ICP-MS when the sediment could not be observed. 2.3.3
Obturator digestion (OD)
One milliliter of hepatocyte solution and 2.0 ml concentrated HNO3 were added into a PTFE bomb. After 10 min, the bombs were sealed off and put into an oven at 80 °C for 2 h. Afterwards, the bombs were cooled down and then placed in the oven at 170 °C for another 2 h. After cooling down the bomb, 200 ȝl H2O2 was added onto the residue inside the bomb to form a clear solution. This solution was diluted to 10 fold for ICP-MS analysis. 2.3.4
Microwave digestion (MD)
Microwave digestion was performed using Speed wave MW-3+ microwave digestion system (Berghof, Germany). One milliliter of hepatocyte solution and 2 ml of concentrated HNO3 were added into a PTFE bomb. The digestion temperature increased as the gradient of 100, –180, –200, and
CAO Xuan et al. / Chinese Journal of Analytical Chemistry, 2009, 37(1): 19–24
–210 °C, every temperature stood for 10 min. After digestion, 200 ȝl of H2O2 was added and allowed to stand for 5 min to enhance the digestion efficiency and to obtain a clear solution. This digestion solution was diluted 10 fold before the analysis. 2.4
Analysis of arsenic speciation
2.4.1
Sample pretreatment
Table 1 Instrumental parameters for total As determination and As speciation analysis Total arsenic ICP-MS RF power (W) RF matching (V) Carrier gas flow rate (l min–1) Peristaltic pump flow rate Monitored signals
Agilent 7500a 1350 1.6 1.14 0.1 rps 75 As, 72Ge, 35Cl, 77Se, 83Kr and 34S
Arsenic speciation
The samples were thawed at 4 °C in 99.99% nitrogen atmosphere, which could prevent the oxidation of arsenic metabolites, and then samples were filtered using 0.45 ȝm membrane for the final analysis. 2.4.2
Chromatography and ICP-MS operating conditions
An anion exchange column (Hamilton PRP-X100) was applied to separate arsenic metabolites in hepatocytes. Both chromatography and ICP-MS operating conditions are listed in Table 1.
3 3.1
Results and discussion Comparison of four AsT methods
To obtain the accurate total element concentration in biological samples, pretreatment was a crucial step. Generally, digestion is the best choice. Digestion includes wet digestion and dry digestion. For AsT, dry digestion could be more efficient, and the operation procedure is simple. However, As could be sublimed or easier to form arsenic dimer and arsenic oxide at very low temperature. These substances escaped and caused arsenic loss in the procedure of dry digestion. Therefore, dry digestion was not suitable for arsenic digestion. The recommended arsenic digestion method was wet digestion. Wet digestion method was performed in an enclosed space. By strong acid and oxidants, the samples were digested to form homogeneous and steady solution. Wet digestion could be classified to obturator digestion, microwave digestion, and cold digestion, etc. For obturator and microwave digestions, the samples were both digested using strong acid in an enclosed space at high temperature. However, there was difference in the heating modes. The former was external heating, that is, the samples in obturator were heated in ovens or muffles to achieve digestion. The latter was internal heating that used the penetrability and activation of microwave to achieve digestion.
HPLC Analytical column Flow rate(ml min–1) Injection volume (μl) Mobile phase A Mobile phase B Gradient program
Agilent 1100 Series Hamilton PRP-X100 (250 mm × 4.1 mm, 10 μm) 1.8 25 H2O 50 mM (NH4)2CO3˄pH=7.4 adjusted by acetic acid˅ Time (min)
A (%)
B (%)
0 15 30
100 0 100
0 100 0
While cold digestion was suitable for the samples, which contain relatively less organics. In cold digestion, the samples were digested without heating but just by the strong acid or oxidants. In this study, these digestion methods were investigated systematically to find out the optimal digestion methods for the hepatocytes. Table 2 shows the comparison results of AsT determination with the four digestion methods. Among these methods, obturator digestion and microwave digestion methods showed the excellent performance than any others. The direct injection and cold digestion methods were inferior. Especially for direct injection, approximately only 50% AsT was obtained compared with microwave digestion. The RSDs of AsT measurement with obturator digestion and microwave digestion methods were lower than that of the others, whereas the highest was obtained when using direct injection. This suggested that the cells were not broken up thoroughly or samples were not well homogenized after ultrasonication, which may result in the poor accuracy and stability of AsT determination with direct injection. The RSD% of AsT determination with cold digestion method was modest; cold digestion could enhance the homogeneity of the samples, however, compared with heating digestion (obturator and microwave digestion), and the accuracy and stability of the determination with cold digestion could not meet the need of analytical chemistry. There was no obvious difference in the results from Table 2.
Table 2 Comparison of 4 total arsenic determination methods (ȝg l–1) Cultured conca Blank 1 ȝM group 5 ȝM group 10 ȝM group 20 ȝM group a
DJ 2.01 ± 1.99 12.2 ± 7.2 24.4 ± 9.3 50.0 ± 12 98.2 ± 9.9
CD 2.31 ± 1.60 14.5 ± 5.3 33.1 ± 4.1 78.8 ± 8.2 110.3 ± 8.1
MD 2.64 ± 0.34 18.1 ± 0.7 48.2 ± 1.3 99.8 ± 2.7 154.0 ± 2.1
OD 2.75 ± 0.49 17.7 ± 1.0 48.8 ± 1.2 101.8 ± 3.8 156.2 ± 3.2
mean ± standard deviation, n = 3; DJ: represents direct injection; CD: represents cold digestion; MD: represents microwave digestion; OD: represents obturator digestion.
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With both the methods of obturator digestion and microwave digestion nevertheless, obturator digestion consume relatively long time, which might increase the possibility of pollution. In this study, the microwave digestion was chosen to treat the sample prior to the determination of AsT in Chang liver hepatocytes. 3.2
Interferences elimination
The interference was the main obstacle to interfere the accuracy of arsenic determination. There are two types of interferences in ICP-MS, mass spectroscopic and nonmass spectroscopic interferences. Mass spectroscopic interference of arsenic was derived from 35Cl, which was abundant in biological samples. The isotope of 35Cl could combine with carrier gas (40Ar) in plasma to form 40Ar35Cl, and a quadrupole mass filter could not distinguish 75As and 40Ar35Cl. To solve this problem, the samples were diluted to decrease the concentration of 35Cl, and meanwhile, ICP-MS operating conditions were optimized and interference equation 75As = 75 M – 77M (3.127) + 82M (2.733) – 83M (2.757) was used to resolve this polyatomic interference. Nonmass spectroscopic interferences was mainly derived from sample matrix, and high matrix of biological samples would suppress the signals of arsenic. In this study, the isotope of 89Y was used as an internal standard to adjust this suppression. 3.3
Linear range and method precision
A series of 0–200 ȝg l–1 arsenic standard solutions was prepared for the calibration curve. The excellent linear correlation coefficient was obtained (0.9998). Dual mode detector and nine order of dynamic range of ICP-MS confirmed arsenic determination validation. The measurement precision was evaluated by determination of 1 ȝM arsenic group cells using four different methods of sample pretreatment for seven times. The RSDs of the measurement with each method were listed in Table 3.
Compared with direct injection and cold digestion, the RSDs of obturator and microwave digestion methods were superior, which were 1.21% and 2.14%, respectively. Besides, long-term detection precision was also investigated in six days (one time determination each day), and the results of interday precision of six days are also listed in Table 3. The best long term detection precision could be obtained with Microwave digestion method, while the worst precision was observed with using direct injection (RSD = 12.6%). Compared with obturator method, microwave digestion shows the preferable performance in long-term determination, although its day precision was slightly inferior to obturator digestion. 3.4
Detection limits and spike recovery
The detection limits were confirmed by 3 × SD (Standard Deviation) of eleven times independent determination of blank sample, and the results are shown in Table 4. The accuracy of the method for the As determination was assessed by the spike recovery because of the lack of cell-certificated relative materials (CRMs). Approximately 100% background arsenic concentration was spiked into 5 ȝM arsenic group cells. After those of four pretreatment methods, recovered spike concentrations were determined to calculate the recovery ratio, which are shown in Table 4. The spike recoveries ranged from 84.4%–108.1%, which indicate that the method accuracy is quite satisfactory except using the direct injection method (64.0%). 3.5
Arsenic speciation in Chang liver hepatocytes
Inorganic arsenic could be metabolized to yield monomethylated, dimethylated metabolites, and possibly some intermediate arsenic motablites. This process was termed as biomethylation, which is a major metabolic pathway for inorganic arsenic in humans and in most animal species[6–8], although the detailed process was not well understood yet.
Table 3 Measurement precision and inter-day precision Method DJ CD MD OD
Precision (RSD, %)
Detection limit (ȝg l–1)
1st day (ȝg l–1)
2nd day (ȝg l–1)
3rd day (ȝg l–1)
4th day (ȝg l–1)
5th day (ȝg l–1)
6th day (ȝg l–1)
Inter-day precision (RSD, %)
7.80 5.60 2.14 1.21
0.93 0.74 0.91 0.79
12.3 14.6 17.9 17.6
9.40 13.3 18.3 17.5
11.5 15.6 18.0 17.8
10.5 15.8 18.4 18.0
8.70 12.0 18.2 17.0
10.4 13.1 18.4 17.4
12.6 10.9 1.15 1.97
DJ: represents direct injection; CD: represents cold digestion; MD: represents microwave digestion; OD: represents obturator digestion.
Table 4 Detection limits and spike recovery Method DJ CD MD OD
Detection limits (ȝg l–1) 0.93 0.74 0.91 0.79
Spike recovery Original content (ȝg l–1) 24.4 35.7 48.9 49.2
Added (ȝg l–1) 20 30 40 40
Found (ȝg l–1) 28.4 55.3 96.1 88.4
Recovery (%) 64.0 84.1 108.1 95.7
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Biomethylation mainly occurred in liver[9,10], and the determination and distinguishing of those biomethylation metabolites would reflect the degree of arsenic metabolism in the organism. Furthermore, the qualitative characterization of methylated arsenic metabolites in liver might give intuitive support to uncover biomethylation mechanism. In this study, an anion exchange chromatography was used for the separation of six arsenic species, such as As(III), As(V), DMA, MMA, AsB, and AsC. Deionized water (A) and 50 mM (NH4)2CO3 (B) were used as the mobile phases. The grade elution conditions are listed in Table 1, and Fig.1 showed the HPLC-ICP-MS spectrum of those six arsenic species. All these arsenic species referred in this study could be baseline separation using this chromatography conditions. Using these chromatography conditions, arsenic species in 1.0 ȝM arsenic group Chang liver hepatocytes were analyzed. To prevent the interference of 40Ar and 35Cl[10], the signals of 75 As, 72Ge, 35Cl, 77Se and 83Kr were monitored simultaneously. The arsenic species in Chang liver hepatocytes are illustrated in Fig.2. Among all the species, As(V) was found to be the main component in hepatocytes. The observed As(V) possibly be derived from the oxidation of As(III), which was not metabolized by hepatocytes. Besides As(V), As(III), DMA, and an unknown arsenic peak were also found in hepatocytes. As we know, the arsenic metabolism in human liver was not well understood so far. But arsenic biomethylation did exist in liver when inorganic arsenics were ingested into human. As(V) was first transformed to As(III) and then methylated to MMA; MMA, which was a mark of first step methylation, could be methylated again to DMA through a series of intermediate arsenic metabolites. These two steps of methylation had been confirmed elsewhere[11,12]. As shown in Fig.2, the finding of DMA showed that the methylation did occur in Chang liver hepatocytes. Interestingly, instead of MMA, an unknown arsenic peak was also found in the chromatogram. It deduced that this unknown peak might be intermediate metabolites of MMA because MMA was not observed in the chromatogram. To prove this hypothesis, an appropriate content of H2O2 was added into the samples and then made stable for 4 h. Arsenic species in hepatocytes after H2O2 treatment are shown in Fig.3. Compared to Fig.2, both As(III) and the unknown peak were disappeared. As a counterpart, the peak area of As(V) increased about 25%, which is resulted from the oxidation of As(III). Meanwhile, MMA was observed. There are some literatures[13], indicating that H2O2 could break the bond between the arsenic and sulfhydryl group. Therefore, this phenomenon testified that the unknown peak was the intermediate metabolites of MMA. This unknown arsenic species possessed significant importance owing to the identification of arsenic intermediate metabolites that were the key to explain arsenic metabolism in the human body[11,12]. If this arsenic species could be identified exactly, it might give a strong support to well understand the arsenic metabolism in human body.
Fig.1 HPLC-ICP-MS spectrum of 6 mixed As species standards 1. AsC; 2. AsB; 3. As(III); 4. DMA; 5. MMA; 6. As(V)
Fig.2
HPLC-ICP-MS chromatographic spectrum of As species compounds in 1.0 ȝM group
Fig.3
HPLC-ICP-MS chromatographic spectrum of As species compounds in 1.0 ȝM group after H2O2 treatment
4
Conclusion
The effects of three digestion methods (cold digestion, obturator digestion, and microwave digestion) on the ICP-MS determination of arsenic in Chang liver hepatocytes after ultrasonic disintegration were systematically studied. The comparison results indicate that the better performance on the arsenic determination in Chang liver hepatocytes, including digestion efficiency, detection precision, interday precision, and spike recovery, could be expected with obturator and microwave digestions. However, when digestion time of samples was taken into account, microwave digestion was
CAO Xuan et al. / Chinese Journal of Analytical Chemistry, 2009, 37(1): 19–24
finally recommended as the best method to determine the arsenic in Chang liver hepatocytes. Furthermore, a HPLC-ICP/MS hyphenated technique was established and then used to analyze the arsenic species in Chang liver cells. An unknown arsenic intermediate metabolite was found, which might supply some useful information to well understand arsenic metabolism in human body.
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