Background: Stability of chemical speciation during sample handling and storage is a prerequisite... more Background: Stability of chemical speciation during sample handling and storage is a prerequisite to obtaining reliable results of trace element speciation analysis. There is no comprehensive information on the stability of common arsenic species, such as inorganic arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid, dimethylarsinic acid, and arsenobetaine, in human urine. Methods: We compared the effects of the following storage conditions on the stability of these arsenic species: temperature (25, 4, and −20 °C), storage time (1, 2, 4, and 8 months), and the use of additives (HCl, sodium azide, benzoic acid, benzyltrimethylammonium chloride, and cetylpyridinium chloride). HPLC with both inductively coupled plasma mass spectrometry and hydride generation atomic fluorescence detection techniques were used for the speciation of arsenic. Results: We found that all five of the arsenic species were stable for up to 2 months when urine samples were stored at 4 and −20 °C without...
Toxicological sciences : an official journal of the Society of Toxicology, Jan 9, 2015
Arsenic is a proven human carcinogen and associated with a myriad of other adverse health effects... more Arsenic is a proven human carcinogen and associated with a myriad of other adverse health effects. This metalloid is methylated in human liver to monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), dimethylarsinic acid (DMA(V)), and dimethylarsinous acid (DMA(III)) and eliminated predominantly in urine. Hepatic basolateral transport of arsenic species is ultimately critical for urinary elimination; however, these pathways are not fully elucidated in humans. A potentially important human hepatic basolateral transporter is the ATP-binding cassette (ABC) transporter multidrug resistance protein 4 (MRP4/ABCC4) that in vitro is a high affinity transporter of DMA(V) and the diglutathione conjugate of MMA(III) [MMA(GS)2]. In rats, the related canalicular Mrp2/Abcc2 is required for biliary excretion of arsenic as As(GS)3 and MMA(GS)2. The current study used sandwich cultured human hepatocytes (SCHH) as a physiological model of human arsenic hepatobiliary transport. Arsenic ...
Human exposure to high concentrations of arsenic from water and food is an important health conce... more Human exposure to high concentrations of arsenic from water and food is an important health concern. Although 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone) was used for more than 60 years as a feed additive to feed chickens, little is known about the metabolism of this arsenic species in chicken. Determination of potential arsenic metabolites present at trace concentrations is an analytical challenge, requiring efficient separation and sensitive detection. The primary objective of this research is to develop a method that enables the identification and quantification of various arsenic species in chicken liver. This report describes a method of high performance liquid chromatography (HPLC) separation with both inductively coupled plasma mass spectrometry (ICPMS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection. Anion exchange HPLC enabled the separation of Roxarsone and other arsenic species within 12 min. Detection with both ICPMS and ESI-MS/MS allowed f...
Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in the drinki... more Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in the drinking water is carcinogenic to the urinary bladder of humans. The highly reactive trivalent organic arsenicals dimethylarsinous acid (DMA(III)) and monomethylarsonous acid (MMA(III)) are formed during the metabolism of inorganic arsenic in vivo in addition to the corresponding mono-, di- and trimethylated pentavalent arsenicals. The objective of this study was to determine if combining arsenicals was additive or synergistic toward inducing cytotoxicity in a rat urothelial cell line. The MYP3 cell line, an immortalized but not transformed rat urinary bladder epithelial cell line, was seeded into appropriate culture wells. Treatment with the arsenicals was begun 24 h after seeding and continued for 3 days. Combinations of arsenicals used were DMA(III) with arsenite, dimethylarsinic acid (DMA(V)) or trimethylarsine oxide (TMAO). Combinations of concentrations used were the LC50, one-quarter or one-half the LC50 of one arsenical with one-half or one-quarter the LC50 of the other arsenical. To determine if MYP3 cells metabolize arsenicals, cells were treated with arsenate, arsenite and MMA(V) as described above and the medium was analyzed by HPLC-ICPMS to determine species and quantity of arsenicals present. When cells were treated with one-quarter or one-half the LC50 concentration of both arsenicals, the cytotoxicity was approximately the same as when cells were treated with half the LC50 concentration or the LC50 concentration, respectively, of either arsenical. Treatment with one-quarter the LC50 concentration of one arsenical plus the LC50 concentration of a second arsenical had similar cytotoxicity as treatment with the LC50 concentration of either of the arsenicals. Quantitation and speciation of arsenicals in the cell culture medium showed that MYP3 cells have some reductase activity but the cells do not methylate arsenicals. The effect on the cytotoxicity of arsenicals in combination was additive rather than synergistic toward a rat urothelial cell line.
Folate binding protein-2 (Folbp2(-/-)) knockout mice have been previously shown to be highly susc... more Folate binding protein-2 (Folbp2(-/-)) knockout mice have been previously shown to be highly susceptible to the teratogenic effects of arsenic. Arsenic biotransformation is achieved primarily by biomethylation. Given the potential close relationship between folate biochemistry and arsenic biotransformation, the aims of our study were to: (1) test whether Folbp2(-/-) mice have altered arsenic biotransformation which would suggest a potential mechanism for their enhanced susceptibility; (2) examine whether dietary folate deficiency alters arsenic biotransformation. Folbp2(-/-) mice were found to have slightly lower plasma folate levels than wildtype mice. No genotype-specific effects were observed in arsenic speciation thereby negating altered biotransformation of arsenic as the mechanism of the enhanced teratogenicity seen in Folbp2(-/-) mice. Reduction in excretion of organic arsenicals was observed during folate deficiency, suggesting an important role for folic acid homeostasis in arsenic biotransformation.
Background: Determination of arsenic species in saliva is potentially useful for biomonitoring of... more Background: Determination of arsenic species in saliva is potentially useful for biomonitoring of human exposure and studying arsenic metabolism. Arsenic speciation in saliva has not been reported previously. Methods: We separated arsenic species in saliva using liquid chromatography (LC) and quantified them by inductively coupled plasma mass spectrometry. We further confirmed the identities of arsenic species by LC coupled with electrospray ionization tandem mass spectrometry. These methods were successfully applied to the determination of arsenite (AsIII), arsenate (AsV), and their methylation metabolites, monomethylarsonic acid (MMAV), and dimethylarsinic acid (DMAV), in >300 saliva samples collected from people who were exposed to varying concentrations of arsenic. Results: The mean (range) concentrations (μg/L) in the saliva samples from 32 volunteers exposed to background levels of arsenic were AsIII 0.3 [not detectable (ND) to 0.7], AsV 0.3 (ND to 0.5), MMAV 0.1 (ND to 0.2...
Arsenic has been used successfully in clinical trials for treating acute promyelocytic leukemia (... more Arsenic has been used successfully in clinical trials for treating acute promyelocytic leukemia (APL). Although sublethal doses of inorganic arsenic are used, little is known about the pharmacokinetics and metabolism of the high levels of arsenic in APL patients. To fill this important gap, this study describes the speciation of arsenic in urine from four APL patients treated with arsenic. Each patient was injected daily with an arsenite (As(III)) solution that contained 10 mg of As(2)O(3) precursor. Speciation analysis of the patient urine samples collected consecutively for 48 h, encompassing two intravenous injections of arsenic, revealed the presence of monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), monomethylarsonic acid (MMA(V)), and dimethylarsinic acid (DMA(V)). The intermediate methyl arsenic metabolites, MMA(III) and DMA(III), were detected in most urine samples from all of the patients when a preservative, diethyldithiocarbomate, was added to the urine samples to stabilize these trivalent arsenic species. The major arsenic species detected in the urine samples from the patients were As(III), MMA(V), and DMA(V), accounting for >95% of the total arsenic excreted. The relative proportions of As(III), As(V), MMA(V), and DMA(V) in urine samples collected 24 h after the injections of As(III) were 27.6 +/- 6.1, 2.8 +/- 2.0, 22.8 +/- 8.1, and 43.7 +/- 13.3%, respectively. The relatively lower fraction of the methylated arsenic species in these APL patients under arsenic treatment as compared with that from the general population exposed to much lower levels of arsenic suggests that the high levels of As(III) inhibit the methylation of arsenic (inhibits the formation of methyl arsenic metabolites). The arsenic species excreted into the urine accounted for 32-65% of the total arsenic injected. These results suggest that other pathways of excretion, such as through the bile, may play an important role in eliminating (removing) arsenic from the human body when challenged by high levels of As(III).
Background: Stability of chemical speciation during sample handling and storage is a prerequisite... more Background: Stability of chemical speciation during sample handling and storage is a prerequisite to obtaining reliable results of trace element speciation analysis. There is no comprehensive information on the stability of common arsenic species, such as inorganic arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid, dimethylarsinic acid, and arsenobetaine, in human urine. Methods: We compared the effects of the following storage conditions on the stability of these arsenic species: temperature (25, 4, and −20 °C), storage time (1, 2, 4, and 8 months), and the use of additives (HCl, sodium azide, benzoic acid, benzyltrimethylammonium chloride, and cetylpyridinium chloride). HPLC with both inductively coupled plasma mass spectrometry and hydride generation atomic fluorescence detection techniques were used for the speciation of arsenic. Results: We found that all five of the arsenic species were stable for up to 2 months when urine samples were stored at 4 and −20 °C without...
Toxicological sciences : an official journal of the Society of Toxicology, Jan 9, 2015
Arsenic is a proven human carcinogen and associated with a myriad of other adverse health effects... more Arsenic is a proven human carcinogen and associated with a myriad of other adverse health effects. This metalloid is methylated in human liver to monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), dimethylarsinic acid (DMA(V)), and dimethylarsinous acid (DMA(III)) and eliminated predominantly in urine. Hepatic basolateral transport of arsenic species is ultimately critical for urinary elimination; however, these pathways are not fully elucidated in humans. A potentially important human hepatic basolateral transporter is the ATP-binding cassette (ABC) transporter multidrug resistance protein 4 (MRP4/ABCC4) that in vitro is a high affinity transporter of DMA(V) and the diglutathione conjugate of MMA(III) [MMA(GS)2]. In rats, the related canalicular Mrp2/Abcc2 is required for biliary excretion of arsenic as As(GS)3 and MMA(GS)2. The current study used sandwich cultured human hepatocytes (SCHH) as a physiological model of human arsenic hepatobiliary transport. Arsenic ...
Human exposure to high concentrations of arsenic from water and food is an important health conce... more Human exposure to high concentrations of arsenic from water and food is an important health concern. Although 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone) was used for more than 60 years as a feed additive to feed chickens, little is known about the metabolism of this arsenic species in chicken. Determination of potential arsenic metabolites present at trace concentrations is an analytical challenge, requiring efficient separation and sensitive detection. The primary objective of this research is to develop a method that enables the identification and quantification of various arsenic species in chicken liver. This report describes a method of high performance liquid chromatography (HPLC) separation with both inductively coupled plasma mass spectrometry (ICPMS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection. Anion exchange HPLC enabled the separation of Roxarsone and other arsenic species within 12 min. Detection with both ICPMS and ESI-MS/MS allowed f...
Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in the drinki... more Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in the drinking water is carcinogenic to the urinary bladder of humans. The highly reactive trivalent organic arsenicals dimethylarsinous acid (DMA(III)) and monomethylarsonous acid (MMA(III)) are formed during the metabolism of inorganic arsenic in vivo in addition to the corresponding mono-, di- and trimethylated pentavalent arsenicals. The objective of this study was to determine if combining arsenicals was additive or synergistic toward inducing cytotoxicity in a rat urothelial cell line. The MYP3 cell line, an immortalized but not transformed rat urinary bladder epithelial cell line, was seeded into appropriate culture wells. Treatment with the arsenicals was begun 24 h after seeding and continued for 3 days. Combinations of arsenicals used were DMA(III) with arsenite, dimethylarsinic acid (DMA(V)) or trimethylarsine oxide (TMAO). Combinations of concentrations used were the LC50, one-quarter or one-half the LC50 of one arsenical with one-half or one-quarter the LC50 of the other arsenical. To determine if MYP3 cells metabolize arsenicals, cells were treated with arsenate, arsenite and MMA(V) as described above and the medium was analyzed by HPLC-ICPMS to determine species and quantity of arsenicals present. When cells were treated with one-quarter or one-half the LC50 concentration of both arsenicals, the cytotoxicity was approximately the same as when cells were treated with half the LC50 concentration or the LC50 concentration, respectively, of either arsenical. Treatment with one-quarter the LC50 concentration of one arsenical plus the LC50 concentration of a second arsenical had similar cytotoxicity as treatment with the LC50 concentration of either of the arsenicals. Quantitation and speciation of arsenicals in the cell culture medium showed that MYP3 cells have some reductase activity but the cells do not methylate arsenicals. The effect on the cytotoxicity of arsenicals in combination was additive rather than synergistic toward a rat urothelial cell line.
Folate binding protein-2 (Folbp2(-/-)) knockout mice have been previously shown to be highly susc... more Folate binding protein-2 (Folbp2(-/-)) knockout mice have been previously shown to be highly susceptible to the teratogenic effects of arsenic. Arsenic biotransformation is achieved primarily by biomethylation. Given the potential close relationship between folate biochemistry and arsenic biotransformation, the aims of our study were to: (1) test whether Folbp2(-/-) mice have altered arsenic biotransformation which would suggest a potential mechanism for their enhanced susceptibility; (2) examine whether dietary folate deficiency alters arsenic biotransformation. Folbp2(-/-) mice were found to have slightly lower plasma folate levels than wildtype mice. No genotype-specific effects were observed in arsenic speciation thereby negating altered biotransformation of arsenic as the mechanism of the enhanced teratogenicity seen in Folbp2(-/-) mice. Reduction in excretion of organic arsenicals was observed during folate deficiency, suggesting an important role for folic acid homeostasis in arsenic biotransformation.
Background: Determination of arsenic species in saliva is potentially useful for biomonitoring of... more Background: Determination of arsenic species in saliva is potentially useful for biomonitoring of human exposure and studying arsenic metabolism. Arsenic speciation in saliva has not been reported previously. Methods: We separated arsenic species in saliva using liquid chromatography (LC) and quantified them by inductively coupled plasma mass spectrometry. We further confirmed the identities of arsenic species by LC coupled with electrospray ionization tandem mass spectrometry. These methods were successfully applied to the determination of arsenite (AsIII), arsenate (AsV), and their methylation metabolites, monomethylarsonic acid (MMAV), and dimethylarsinic acid (DMAV), in >300 saliva samples collected from people who were exposed to varying concentrations of arsenic. Results: The mean (range) concentrations (μg/L) in the saliva samples from 32 volunteers exposed to background levels of arsenic were AsIII 0.3 [not detectable (ND) to 0.7], AsV 0.3 (ND to 0.5), MMAV 0.1 (ND to 0.2...
Arsenic has been used successfully in clinical trials for treating acute promyelocytic leukemia (... more Arsenic has been used successfully in clinical trials for treating acute promyelocytic leukemia (APL). Although sublethal doses of inorganic arsenic are used, little is known about the pharmacokinetics and metabolism of the high levels of arsenic in APL patients. To fill this important gap, this study describes the speciation of arsenic in urine from four APL patients treated with arsenic. Each patient was injected daily with an arsenite (As(III)) solution that contained 10 mg of As(2)O(3) precursor. Speciation analysis of the patient urine samples collected consecutively for 48 h, encompassing two intravenous injections of arsenic, revealed the presence of monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), monomethylarsonic acid (MMA(V)), and dimethylarsinic acid (DMA(V)). The intermediate methyl arsenic metabolites, MMA(III) and DMA(III), were detected in most urine samples from all of the patients when a preservative, diethyldithiocarbomate, was added to the urine samples to stabilize these trivalent arsenic species. The major arsenic species detected in the urine samples from the patients were As(III), MMA(V), and DMA(V), accounting for >95% of the total arsenic excreted. The relative proportions of As(III), As(V), MMA(V), and DMA(V) in urine samples collected 24 h after the injections of As(III) were 27.6 +/- 6.1, 2.8 +/- 2.0, 22.8 +/- 8.1, and 43.7 +/- 13.3%, respectively. The relatively lower fraction of the methylated arsenic species in these APL patients under arsenic treatment as compared with that from the general population exposed to much lower levels of arsenic suggests that the high levels of As(III) inhibit the methylation of arsenic (inhibits the formation of methyl arsenic metabolites). The arsenic species excreted into the urine accounted for 32-65% of the total arsenic injected. These results suggest that other pathways of excretion, such as through the bile, may play an important role in eliminating (removing) arsenic from the human body when challenged by high levels of As(III).
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