Article pubs.acs.org/crt
Identification of Methylated Dithioarsenicals in the Urine of Rats Fed with Sodium Arsenite Baowei Chen,*,†,‡,∥ Xiufen Lu,‡ Lora L. Arnold,§ Samuel M. Cohen,§ and X. Chris Le*,‡ †
MOE Key Laboratory of Aquatic Product Safety, School of Marine Sciences, Sun Yat-Sen University, Guangzhou 510275, People’s Republic of China ‡ Analytical and Environmental Toxicology Division, Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta T6G 2G3, Canada § Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-3135, United States ∥ South China Sea Resource Exploitation and Protection Collaborative Innovation Center, Sun Yat-Sen University, Guangzhou 510275, People’s Republic of China S Supporting Information *
ABSTRACT: Biotransformation of inorganic arsenic results in the formation of methylarsenicals of both oxygen and sulfur analogues. Aiming to improve our understanding of metabolism of inorganic arsenic in animals, we conducted an animal feeding study with an emphasis on identifying new arsenic metabolites. Female F344 rats were given 0, 1, 10, 25, 50, and 100 μg/g of arsenite (iAsIII) in the diet. Arsenic species in rat urine were determined using high performance liquid chromatography (HPLC) separation and inductive coupled plasma mass spectrometry (ICPMS) and electrospray ionization tandem mass spectrometry (ESI MS/MS) detection. Nine arsenic species were detected in the urine of the iAsIII-dosed rats. Seven of these arsenic species were consistent with previous reports, including iAsIII, arsenate, monomethyarsonic acid, dimethylarsinic acid, trimethylarsine oxide, monomethylmonothioarsonic acid, and dimethylmonothioarsinic acid. Two new methyldithioarsencals, monomethyldithioarsonic acid (MMDTAV) and dimethyldithioarsinic acid (DMDTAV), were identified for the first time in the urine of rats treated with iAsIII. The concentrations of both MMDTAV and DMDTAV in rat urine were dependent on the dosage of iAsIII in diet. The concentration of DMDTAV was approximately 5 times higher than that of MMDTAV. MMDTAV has not been identified in any biological samples of animals, and DMDTAV has not been reported as a metabolite of inorganic arsenic in the rats. The identification of novel methylated dithioarsenicals as metabolites of inorganic arsenic in the rat urine provided further insights into the understanding of the metabolism of arsenic.
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INTRODUCTION
commonly identified as the major arsenic metabolites in biological samples. These pentavalent arsenic species are less toxic than inorganic iAsIII.12−15 However, in the process of forming the pentavalent methyl arsenic species, trivalent methylated arsenicals are also produced and have been detected in human and animal urines. These trivalent methylarsenicals are more potent cytotoxicants than inorganic arsenic.13,16−21 Recently, thio-containing methylated arsenicals have also been identified in various biological samples.22−24 Dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) were detected in human urine and blood samples from people who were exposed to arsenic via different pathways.25−28 In animal studies, monomethylmonothioarsonic acid (MMMTAV), DMMTAV, and DMDTAV were found in hamster urine, and MMMTAV and DMMTAV were found in rat urine and blood.29−31 Nevertheless, the knowledge on
Arsenic is a well-recognized Class I human carcinogen. Epidemiological studies have demonstrated that chronic exposure of humans to high levels of inorganic arsenic via drinking water is linked to skin, bladder, and lung cancers, as well as noncancerous adverse effects such as hypertension, diabetes, and cardiovascular effects.1−5 The toxicities of arsenicals vary with their chemical forms and oxidation states.6 Thus, speciation analysis of arsenicals in the biological samples is especially important to improve our understanding of arsenic metabolism and the underlying mechanism of action responsible for arsenic toxicity. Arsenic naturally occurs in the environment mainly in inorganic forms, including arsenite (iAsIII) and arsenate (iAsV).7,8 In many organisms, inorganic arsenic can be transformed into various organic species through a series of biological processes. For example, inorganic arsenic can be methylated through reduction and oxidative methylation reactions.9−11 Monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) are © XXXX American Chemical Society
Received: May 3, 2016
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DOI: 10.1021/acs.chemrestox.6b00151 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Chemical Research in Toxicology
bladder effects. Fresh diet was supplied to the animals at least once a week. Freshly voided urine was collected from all rats between 7:00 and 9:00 a.m. on the 25th day after commencement of arsenic treatment, as previously described,46 and immediately flash frozen in liquid nitrogen. During study week 5, 24-h urine samples were collected on ice to obtain sufficient volume of urine to identify new dose−responsive arsenic species. Urine was centrifuged at 4 °C and 3000 rpm (1877 g) for 10 min. The supernatant was removed, volume was measured, and samples were frozen at −20 °C. The volume of urine collected over 24 h was between 1.6 and 12.5 mL. Food and water were available to the rats throughout the collection of freshly voided urine and 24-h urine. All samples were shipped on dry ice overnight to Edmonton, Alberta, Canada for arsenic speciation analysis. Arsenic Speciation Analysis Using High Performance Liquid Chromatography (HPLC) with Inductively Coupled Plasma Mass Spectrometry (ICPMS). An Agilent 1100 series HPLC system that comprised a pump, degasser, autosampler, column temperature control, and reversed-phase C18 column (ODS-3, 150 mm × 4.6 mm, 3-μm particle size; Phenomenex, Torrance, CA, USA) was used for the separation of arsenicals.27 An octadecylsilane guard cartridge (4 mm × 3 mm) was mounted before the analytical column. The mobile phase consisted of 5 mM tetrabutylammonium, 5% methanol, and 3 mM malonic acid (pH 5.65). The column was equilibrated with the mobile phase for 0.5 h at a flow rate of 0.8 mL/min before sample injection. A 50 μL aliquot of the urine samples was injected, and separation was performed at a flow rate of 1.2 mL/min; column temperature was maintained at 50 °C. The effluent from the HPLC system was directly introduced into the nebulizer of a 7500ce ICPMS (Agilent Technologies, Japan) using PEEK tubing. The ICP was operated at a radio frequency power of 1550 W, and the argon carrier gas flow rate was 0.9−1.0 L/min. The collision cell of the ICPMS instrument was operated in helium mode to reduce isobaric and polyatomic interference. Helium (3.5 mL/min) was used in the octopole reaction cells. Arsenic was monitored at m/z (mass to charge) 75. Chromatograms from HPLC separation and ICPMS detection were recorded by ICPMS ChemStation (Agilent Technologies, Santa Clara, CA, USA). Determination of MMDTAV and DMDTAV Using HPLC Coupled with Electrospray Ionization Tandem Mass Spectrometry (ESI MS/MS). For optimization of operating conditions, synthesized MMDTAV and DMDTAV standards (500 ng/mL as arsenic) dissolved in methanol/water solution (v:v, 1:1) were infused into a triple quadrupole linear ion trap mass spectrometer (4000 QTRAP, MDS Sciex, Concord, ON, Canada) equipped with an electrospray ion source. The ESI was operated in negative ionization mode. The characteristic multiple reaction monitoring (MRM) ion transitions of MMDTAV were 171 → 107, 171 → 123, 171 → 139, and 171 → 156, and the ion transitions of DMDTAV were 169 → 107, 169 → 139, and 169 → 154. The optimal parameters were as follows: ionspray voltage, −4800 V; temperature, 600 °C; curtain gas, 50 L/min. Tables S1 and S2 in the Supporting Information (SI) show MRM ion transition-specific parameters of MMDTAV and DMDTAV, respectively. The ESI MS/MS instrument was coupled with two types of liquid chromatography: the reversed-phase column (ODS-3, 100A, 30 mm × 4.6 mm, 3-μm particle size; Phenomenex, Torrance, CA, USA) was run with a mobile phase containing 5% methanol at a flow rate of 1.0 mL/min, and anion exchange analytical Guard Column Replacement Cartridges (PRP-X100 Analytical Guard Column Replacement Cartridges, 20 × 2 mm, 10-μm particle size, Hamilton, USA) were run with a mobile phase consisting of 20 mM ammonium bicarbonate and 5% methanol (pH 8.0) at a flow rate of 1.0 mL/min. In order to enhance electrospray ionization of methylated dithioarsenicals in negative mode, a postcolumn solution of methanol and ammonium hydroxide (NH4OH) was introduced to mix with the HPLC effluent using a T-joint and splitter. A 50 μL aliquot of urine samples was injected. Following the column, the effluent was split down to 100 μL/min and mixed at the T-joint with methanol (0.6% NH4OH) that was introduced at a flow rate of 100 μL/min. The combined solution flow was continuously introduced to the ESI MS/MS instrument.
where and how these thio-containing arsenicals are produced in the organisms is limited. Incubation of arsenicals (iAsV, DMAV, and trimethylarsine oxide (TMAOV)) with anaerobic microflora in mouse cecum can lead to the formation of thiocontaining arsenicals.32−34 Thioarsenicals were also formed from the incubation of human colon microbiota with inorganic arsenic or with arsenic-contaminated urban soils.35 Thiocontaining arsenicals were also found in the incubation system of dimethylarsinous acid (DMAIII) and human red blood cells (RBCs).36 Several toxicity tests demonstrated that the toxicities of pentavalent thioarsenicals are comparable to those of trivalent oxygen-containing analogues and are higher than those of pentavalent oxygen-containing counterparts.25,30,37−39 The distributions of thio-containing arsenicals were different from those of their oxygen-containing counterparts in the rat and hamster.40,41 Although many efforts have been made to understand the metabolism of arsenic, the metabonomics of arsenic still has yet to be fully elucidated.42 In principle, substituting different numbers of oxygen with sulfur in the methylarsenicals would lead to many thioarsenicals. However, these possible thioarsenicals have not yet been detected in all tested organisms. The main objective of this study is to improve our understanding of biotransformation of inorganic arsenic by identifying new thioarsenicals.
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EXPERIMENTAL SECTION
Materials and Reagents. Stock solutions (1000 mg/L as arsenic) were prepared by dissolving appropriate amounts of iAsIII (SigmaAldrich, St. Louis, MO), iAsV (Sigma-Aldrich, St. Louis, MO), MMAV (Chem Service), DMAV (Sigma-Aldrich), and TMAOV (Tri Chemical Laboratories Inc.) in deionized water (DIW). Working solutions were prepared daily by serial dilutions of stock solutions. MMMTAV and DMMTAV were synthesized according to the methods in the literature.29,43 MMDTAV and DMDTAV were prepared by the reactions of MMAV and DMAV with hydrogen sulfide (H2S). Specifically, DMAV or MMAV and sodium hydroxide (NaOH) at a molar ratio of 1:1 were dissolved in boiling ethanol, and then H2S was bubbled into the boiling solution for 30 min. A white solid was produced after cooling and isolated by filtration. Tetrabutylammonium hydroxide (Sigma-Aldrich), malonic acid (Sigma-Aldrich), ammonium bicarbonate (Sigma-Aldrich), ammonium hydroxide (Fisher), and HPLC grade methanol (Fisher) were used to prepare the mobile phase for the separation of arsenicals. The mobile phase was filtered through a 0.45-μm membrane. A certified reference material (CRM No.18, Human Urine, Japan) was analyzed in parallel with samples for quality control purposes. Our analysis showed that DMAV concentration in the CRM No.18 urine was 38 ± 4 μg/L, which was consistent with the certified value of DMAV (36 ± 9 μg/L). Animal Experiments. Animal experiments were described in our previous publication.30 Briefly, adult female F344 rats 7 weeks old were purchased from Charles River Breeding Laboratories (Raleigh, NC). Female rats were used because they appear to be more sensitive than male rats to the effects of inorganic arsenic.44 The rats were housed in polycarbonate cages (five/cage) on dry corncob bedding in a room with a targeted temperature of 22 °C, humidity of 50%, and a 12 h light/dark cycle, and fed a basal diet (Certified Purina 5002, Dyets Inc., Bethlehem, PA). Food and tap water were available ad libitum throughout the study. Rats were quarantined for 7 days before starting arsenic treatment. Approximately eight-week-old rats were randomized into six treatment groups using a weight stratification method.45 Rats were randomized into 6 groups of 10 rats each. Inorganic AsIII was spiked into the diet at dosages of 0, 1.73, 17.3, 43.3, 86.5, and 173 mg/kg to achieve arsenic doses of 0, 1, 10, 25, 50, and 100 mg/kg, respectively. Doses were based on previous experiments demonstrating the dose−response for urinary bladder effects of dietary inorganic arsenic.44 Similar doses administered in drinking water caused similar B
DOI: 10.1021/acs.chemrestox.6b00151 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
Article
Chemical Research in Toxicology Accurate Mass Determination of MMDTAV and DMDTAV Standards. A quadrupole time-of-flight (Q-TOF) mass spectrometer (QSTAR Pulsar-i hybrid, MDS Sciex, Concord, ON, Canada) was used to measure the accurate mass of synthesized MMDTAV and DMDTAV
standards. Standard solutions (500 ng/mL as arsenic) were prepared in a methanol/water (v:v, 1:1) solution and were introduced into a nanospray ion source using a syringe infusion pump. The nanospray ionization was operated in negative mode. Operation parameters were as follows: ionspray voltage, −3500 V; temperature, 300 °C; ion source gas (1), 50 L/min; ion source gas (2), 50 L/min; curtain gas, 50 L/min; declustering potential (1), −30 V; declustering potential (2), −15 V; focusing potential, −275 V; ion release delay, 38.6 ms; and release width, 16.8 ms. The infusion flow rate was 50 μL/min. The mass accuracy and resolution of the mass spectrometer were checked and calibrated using a polypropylene glycol (PPG) standard tuning solution from Applied Biosystems (CA, USA).
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RESULTS AND DISCUSSION Figure 1a shows a typical HPLC-ICPMS chromatogram of arsenic species in the freshly voided urine samples from a rat receiving iAsIII in the diet. It demonstrated the presence of iAsIII, iAsV, MMAV, DMAV, TMAOV, MMMTAV, and DMMTAV, as well as two unidentified arsenic metabolites (M1 and M2). MMAV, DMAV, TMAOV, MMMTAV, and DMMTAV were determined as the metabolites of iAsIII in the urine of rats that received iAsIII in the diet, as reported in our previous publication.30 In addition to these, two unidentified arsenicals with a longer retention time were also present in rat urine. Although iAsIII and TMAOV coeluted immediately following the solvent, unidentified arsenicals were well separated from other identified species using
Figure 1. Chromatograms obtained from HPLC-ICPMS analyses of a freshly voided urine sample from female F344 rats that received iAsIII in the diet (a) and the 24-h urine sample spiked with either MMDTAV (b) or DMDTAV(c) standards. The 24-h urine sample (solid line); the same sample spiked with the MMDTAV standard (dashed line) (b); and the same sample spiked with the DMDTAV standard (dashed line) (c). For all analyses, a 50-μL aliquot of sample was injected, and arsenic was monitored at m/z 75 using an Agilent 7500ce octopole reaction system ICPMS. Chromatographic separation of arsenicals was performed on a reversed-phase ODS-3 column (150 × 4.6 mm, 3-μm particle size, Phenomenex). The mobile phase contained 5 mM tetrabutylammonium, 3 mM malonic acid, and 5% methanol (pH 5.65), and the flow rate was 1.2 mL/min. The column temperature was maintained at 50 °C.
Figure 2. Mass spectra obtained from ESI MS analyses of synthesized methylated dithioarsenical standards using Q-TOF in negative mode. C
DOI: 10.1021/acs.chemrestox.6b00151 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
Article
Chemical Research in Toxicology
Figure 3. Chromatograms obtained from HPLC-ESI MS/MS analyses of a 24-h urine sample of a female F344 rat that received iAsIII in the diet. The MS/MS analyses focused on four characteristic MRM ion transitions of MMDTAV, 171 → 107, 171 → 123, 171 → 139, and 171 → 156. (a) Reversed-phase chromatography (Phenomenex, reversed phase ODS-3, 30 × 4.6 mm, 3-μm particle size) was used with the mobile phase containing 5% methanol at a flow rate of 1.0 mL/min. (b) Anion exchange chromatography (Hamilton, PRP-X100 Analytical Guard Column Replacement Cartridges, 20 × 2 mm,10-μm particle size) was used with the mobile phase containing 20 mM ammonium bicarbonate and 5% methanol (pH 8.0) at a flow rate of 1.0 mL/min.
To test our hypothesis, we synthesized MMDTAV and DMDTAV standards so that these standards could be used to determine the identity of the unknown arsenicals. Q-TOF mass spectrometry was used to check the accurate mass of the arsenic standards. Figure 2a demonstrates that the MMDTAV standard had three peaks with m/z values of 170.8899 ± 0.0037, 171.8936 ± 0.0031, and 172.8883 ± 0.0022, respectively. These measured values are in good agreement with the theoretical values of MMDTAV: 170.8911, 171.8931, and 172.8879 (mass error
