Stability of monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII) in human urineand mouse tissues

Authors

• Uttam K Chowdhury Department of Molecular and Cellular Biology, The University of Arizona, Life Sciences South, Tucson, Arizona, 85721-0106, USA

Abstract

Urine samples were collected from 75 subjects in theLagunera area of Mexico. There were four groups, based on totalarsenic concentrations in their drinking water(9-100μg/L).After collection,the samples were immediately put in a portable iceboxcontaining dry ice, and they were kept frozen while beingtransported to the University of Arizona, Tucson where theywerestored at -70°C before analysis.Arsenic metabolites,including MMA(III) and DMA(III),were measured in urine samples by usingHPLC-ICP-MS.

       The average percentage of MMA(III) in urine samples of arsenic exposed people in the Lagunera area of Mexico were 0.44%, 0.26%, N.D. (Not Detectable), and 0.20% of total arsenic for the groups 4, 1, 3, and 2 where arsenic concentrations in their drinking water were 9, 17.5, 52, and 100 (µg/L), respectively.This small percentage of MMA(III) were detected only in 14urine samples (~18%)out of total 75 samples. DMA(III) was not measured in any of these urine samples (n=75) but measured DMA(V) in allsamples.The highest percentage of arsenic metabolites was DMA(V) (61% to 74%) of the total arsenic in urine. It indicates thatmost of the MMA(III) methylated to DMA(V) in tissues,butlesspercentageor most of the DMA(V) may be reducedto DMA(III) and due toits instability,itcould be immediately oxidized to DMA(V)in tissues again or in urine after collection. These findingssuggest that the +5-oxidation state of arsenic metabolite, DMA(V), could be themost dominant methylated arsenic metabolite in humans’ urine of arsenic exposed population.

In animal tissues,the +3-oxidation state of arsenic metabolites,MMA(III) and DMA(III),were measured in mice tissues after administering a single intra-muscular dose of sodium arsenate(4.16 mg As/kg body weight).Liver, kidneys, urinary bladder tissue, lungs, testes, and heart were removed at the following times, 0, 0.5, 1, 2, 4, 8, and 12 h.The tissues were homogenized at 4°C (cold room),and the homogenized samples were extracted immediately. After extraction, I measured the arsenic species as soon as possible by using HPLC-ICP-MS(Chowdhury, et al., 2006).

The concentration of the very toxic MMA(III) was significantly higher than that of MMA(V) in all of the tissues tested (Liver, kidneys, urinary bladder tissue, lungs, testes, and heart). At 2 h, after injection, the highest concentration of MMA(III) was in the kidneys.Compared with the other species of arsenic, MMA(V)concentrations were the lowest in all tissues examined.

On the other hand, the concentration of DMA(V) was higher than DMA(III) in the liver,kidneys,urinary bladder tissue, lungs, and testes at all times. DMA(V) accumulated at a higher concentration in the urinary bladder tissue and lungs, but the concentration of DMA(III) was the highest in the urinary bladder tissue, followed by the kidneys, lungs, heart, testes, and liver. In all of the tissues, both DMA(V) and DMA(III) were highest at 4 h for the WT (wildtype) mice. The concentration of DMA(III) was significantly higher in the urinary bladder tissue than in other tissues (p<0.05), except in the kidneys of the mice.

       The results indicate that MMA(V) reduced to MMA(III) faster, comparing to DMA(V) to DMA(III). It could be that most of the MMA(V) reduced to MMA(III) and methylated to DMA(V), but less/most of the MMA(III) converted to DMA(V)↔ DMA(III), but DMA(III) may be very instable and oxidized to DMA(V) very quickly. This might have been because the DMA(III) would be more instable in the tissues, comparing to MMA(III), and oxidized faster, compared to MMA(III). Also, it could be suitable for the bonding of MMA(III) to tissue components or proteins,compared to DMA(III).

These results also indicate that due to the instability and faster oxidation of DMA(III),therewas no detectable level of DMA(III), but low level of MMA(III) was measuredin some urine samples due to its more stability and bonding capacity with proteins,compared to DMA(III). Both MMA(III) and DMA(III) were detected in tissues but not in urines, and these reports have suggested that tissue levels of MMA(III) and DMA(III) are both more relevant and less susceptible to oxidative artifacts than urine samples.

       In conclusion, the distributionof arsenic metabolites in urine was supported with the distribution of these species in mouse tissues.The trivalent MMA(III) compound could be themost dominant,very toxic arsenic metabolite in humans’ tissues fora short period of time (e.g., MMA(III) was highest at 2 h in mouse tissuesafter injectionof sodium arsenate) but not in urine because it methylated to DMA(V) in tissues, and some percentage of MMA(III) could also be oxidized to MMA(V) again. On the other hand, DMA(V), instead of DMA(III), would be another dominant arsenic metabolite in tissues as well as in urine because we found the final metabolites of arsenic in urine where most of the arsenic metabolite was DMA(V).Thestability/instability of MMA(III) and DMA(III)may depend on biological environment, genetic variability, and other factors.However,more experiments are needed to understand the mechanism of inorganic arsenic biotransformationand stability or instability of highly toxic arsenic metabolites, MMA(III) and DMA(III).

Background

The mechanisms of the toxicity and carcinogenicity ofinorganic arsenic at the molecular level in humans remain anenigma^1-8, even though there aremillions of people in the world that are drinking water, containingcarcinogenic concentrations of inorganic arsenic. For example,more than 25 million people in Bangladesh⁹ and 6 million peoplein West Bengal, India⁹, are drinking water that contains arsenicconcentrations above 50 μg/L, eventhough the WHO recommends that arsenic in drinking water should notexceed 10 μg/L. An estimated 36 million people in the BengalDelta are also at risk for arsenic-caused cancer¹⁰. Chronic exposure to inorganic arsenic has led to cancerof the skin, lungs, urinary bladder tissue, kidneys, and liver^11-14.

The chemical forms of arsenic determine the toxicity andbioavailability of arsenic compounds^7,15. Although the tissue distributions of arsenicspecies with an oxidation state of +5 have been investigatedextensively, this is not the case for the more reactive MMA(III) and DMA(III) found in human urine¹⁶.

The studies have demonstrated that when humans havebeen chronically exposed to inorganic arsenic, they excreted inthe urine arsenic bio-transformants in the +3 oxidation state^16,17(Fig 1). These importantstudies have been confirmed by others^18,19. The +3-oxidation state metabolites,MMA(III) and DMA(III), are more potent cytotoxins,genotoxins, and enzyme inhibitors than inorganic arsenate,arsenite, or the +5-oxidation state methylated arsenicmetabolites, MMA(V), and DMA(V)^20-25. High concentrations of MMA(III),monomethylarsine and DMA(III), dimethylarsine damagedDNA in vitro²⁶.Therefore, there is an increasing interest in the determination ofMMA(III) and DMA(III) in studies of arsenic metabolism andhealth effects.

Figure 1. Proposed pathway for inorganic arsenic biotransformation. Abbreviations: SAM, S-adenosyl-L-methionine; SAHC, S-adenosyl-L-homocysteine; GSTO,glutathione-S-transferase-omega; GSH, glutathione.

The detection and quantification of urinary MMA(III) andDMA(III) in various populations have been reported over the past20 years. Some reports suggestthat in exposed populations, these trivalent compounds are majoror even dominant components of urinary arsenic^18,19,27. Other investigators suggest that there islittle or no DMA(III), and very low levels ofMMA(III) occur in populations with a range of arsenicexposures^28-30. Based on these prior reportswith highly variable findings, the usefulness ofmeasuring urinary arsenic metabolites as a biomarker of exposure or susceptibility has been anopen question of importance for planning future arsenic exposed populationstudies.

However, there are a few studies on the oxidative stability of MMA(III) and DMA(III) arsenic species. It is important to know whether and to what extent these trivalent metabolites are changed during sample storage and handling. The objective of this study was to find the distribution of arsenic metabolites, including MMA(III) and DMA(III),in human urine samples where the samples were immediately frozen after collection and kept frozen during transportation from Mexico to the University of Arizona, Tucson. These samples were immediately stored at -70^oC after arrival until analysis. It is also very important to know the oxidative stability of the +3 oxidation state metabolites, MMA(III) and DMA(III), in tissues, and we studied to find the distribution of arsenic metabolites in mice tissues after administering a single intra-muscular dose of sodium arsenate. Information about the concentration and distribution of these +3 arsenic species in tissues might help in elucidating their biotransformation process, stability/instability in tissues, and distribution in urine.

Materials and Methods

1. Arsenic metabolites in human urine of arsenic exposed people

Reagents. The chemicals used and their sources are as follows: Sodium arsenate (Na₃AsO₄; ACS reagent grade) from MCB Reagents(Cincinnati, OH); DMA(V), dimethylarsinicacid [(NaCH₃)₂AsO₂], ammonium phosphate (dibasic), and arsenobetaine from Sigma ChemicalCo. (St. Louis, MO); sodium m-arsenite (NaAsO₂) and ammonium nitrate from Sigma-Aldrich Co. (St. Louis, MO); MMA(V), monomethylarsonic acid/disodium methylarsenate (Na₂CH₃AsO₃) from ChemService, Inc. (West Chester, PA).The source of MMA(III), monomethylarsonous acid and DMA(III), dimethylarsinous acid were the solid oxide (CH₃AsO) and solid iodide[(CH₃)₂AsI], respectively. The stock solution (1000ppm) of MMA(III) and DMA(III) were prepared as follows: MMA(III) in doubly deionized water, but DMA(III) in 10% methanol + doubly deionized water and stored both at -70^oC.Thearsenic standard solution was from SPEX Certiprep (Metuchen,NJ). All other chemicals were analytical reagent grade or the highest quality obtainable. Water was doubly deionized and distilled.

Subjects.Urine samples and blood samples were collected from 75subjects in theLagunera area of Mexico. There were four groups, based on total arsenic concentrations in their drinking water(9-100μg/L).

Urine and blood collection. All collecting containers were soaked overnight in 2% nitric acid (Baker analyzed for trace metal analysis) (J. T. Baker, Inc. Phillipsburg, NJ) and rinsed with double distilled and deionized water. All plastic measuring and collecting equipment were similarly washed, sealed in bags, placed in locked footlockers, and transported by air to the site of the study at the same time as the investigators. After collection, the urine sample was immediately frozen in a portable icebox, containing dry ice. Blood was collected by venous puncture, into Vacutainers containing EDTA, transferred to a vial, and immediately frozen. The samples were kept frozen while being transported to the University of Arizona, Tucson where theywerestored at -70°C before analysis.

Separation techniques of urinary arsenic metabolites. Arsenic concentration in drinking water is the reason for the elevated levels of arsenic in urine. Even arsenic from seafood(arsenobetaine, AsB) may be responsible for the elevated levelsof arsenic in urine. Thus, to know the nature of arseniccontamination and the measurement of arsenic metabolites,HPLC-ICP-MS(High Performance LiquidChromatography- Inductively Coupled Plasma-MassSpectrometry) is the most advanced and reliable technique. The method of Gong et al. (2001)³¹ could not separate AsB and AsBwas overlapped with arsenite (Figs.2B & 2D). In this study, anHPLC-ICP-MS method³² was modified by Chowdhury UK (2021)³³ for the measurement of arsenic metabolites, including AsB in urine(Figs. 2C & 2D). One of the urine samples contained very high levels of arsenic (697 μg/L urine) due to AsB(509 μg/L urine, i.e., 73% of total arsenic; Fig. 2D), and we excluded this sample from our results.

Arsenic metabolites[AsB, As(V), As(IlI), MMA(V), and DMA(V)]analysis. Frozen urine sampleswere thawed at room temperature, filtered with a 0.45μm filter (Nanosep MF Centrifugal Devices, Pall Life Sciences, AnnArbor, MI), and diluted 5-fold using Milli-Q water beforeinjection. An HPLC-ICP-MS speciation method³² was modified³³ for themeasurement of arsenic concentrations. The HPLC system consisted of a PerkinElmer Series 200 HPLC with an anion exchange column (Gemini PRP-X100, 10μm, 250 x 4.6mm,Hamilton Company, Nevada)(Fig 2 and Method 2). The mobile phase (pH 8.5)contained 10 mM ammonium nitrate and 10 mM ammonium phosphate(dibasic) at a flow rate of 1 mL/min. The column temperature was maintained at 30°C. An ELAN DRCe ICP-MS(Perkin-Elmer) with a cyclonic quartz spray chamber, and Meinhard nebulizer was used as a detector for the analysis of arsenic species [AsB, As(V), As(III), MMA(V), and DMA(V)] in urine at 4°C and sample injection volume 50 μL. The operating parameters were as follows:R_fpower, 1400 W; plasma gas flow, 15 L/min; nebulizer gasflow, 0.82 L/min; auxiliary gas flow, 1.2 L/min; oxygen flow forDRC, 0.87 mL/min; and arsenic was measured at miz 91. The working detection limits were 0.80 - 1.75 μg/L forarsenicmetabolites. Accuracy values were calculated by spiking standard compounds of all five species (5 μg/L) in urine samples. The recoveries of the added compounds were 98-103%. Standard samples (5 μg/L), containing all five arsenic species, were also analyzed after analysis of the urine samples each day. The values of mean ± SE for AsB, As(V), As(III), MMA(V),and DMA(V) were found to be 4.86 ± 0.08, 5.09 ± 0.11, 5.16 ± 0.11,5.02 ± 0.10, and 4.90 ± 0.05μg/L, respectively.

The +3 oxidation state metabolites, MMA(III) and DMA(III) analysis.Frozen urine samples were partially thawed at room temperature, then the samples were completely thawed on iceandimmediately filtered with a 0.45 μm filter(Nanosep MF Centrifugal Devices, Pall Life Sciences, AnnArbor, MI) in cold room (4^oC)beforeinjection. An HPLC-ICP-MS speciation method³¹ was used for themeasurement of arsenic concentrations. The HPLC systemconsisted of a PerkinElmer Series 200 HPLC with a reverse-phase column (Gemini 5μm, C18, 110A, 150 x 4.6mm,Phenomenex, Torrance, CA). The mobile phase (pH 5.85)contained 4.7 mM tetra-butyl ammonium hydroxide, 2 mM malonic acid, and 4% (v/v)methanolat a flow rate of 1.2 ml/min (Fig 2 and Method 1). The columntemperature was maintained at 50°C. An ELAN DRCe ICP-MS(Perkin-Elmer) with a cyclonic quartz spray chamber andMeinhard nebulizer was used as a detector for the analysis ofarsenic species [MMA(III) andDMA(III)] in urine at 4°C and sample injection volume 50 μL. The operating parameters were as follows:R_f power, 1400 W; plasma gas flow, 15 L/min; nebulizer gas flow, 0.82 L/min; auxiliary gas flow, 1.2 L/min; oxygen flow forDRC, 0.87 mL/min; and arsenic was measured at miz 91. The detection limits for MMA(III) and DMA(III) were 0.09 and 0.16 μg/L, respectively. Accuracy values were calculated by spiking standard compounds of all six species [1μg/L; As(V)+As(III)+MMA(V)+MMA(III)+DMA(V)+DMA(III)]inurine samples. The recoveries of the added compounds of MMA(III) and DMA(III) were 90% and 97%, respectively. Standard samples (1μg/L) containing all six arsenic species were also analyzed after analysis of the urine samples every day. The values of average ± SD forMMA(III),and DMA(III) were found to be 0.90 ± 0.05 and 0.97 ± 0.27μg/L, respectively.

Total arsenic analysis in urine samples. Urine samples in acid washed polypropylene tubes were digested with nitric acid (5: 1) while a water bath for 40 min at 70°C. Freeze-dried urine reference material for toxic elements containing arsenic at a level of 220 ± 10 μgAs/L was used for quality control and to validate the assay. After acid digestion, analysis of this standard by ICP-MS yielded a range of 216.0 - 236.0 μg As/L with a range of recoveries of 98.18 - 107.27%. We also analyzed the spiking standard compounds of all the arsenic species [AsB, As(V),As(III), MMA(V), and DMA(V)] at levels of 10 μg total As/L and 20 μg total As/L. The recoveries of the spiking samples were 104.20 % (10.42 ± 0.13 μgtotal As/L) and 97.70 %(19.54 ± 0.24 μgtotal As/L), respectively.

Statistical analysis: The mean and standard error (SE) were calculated. The unpaired t test (Graph Pad Software, Inc., 2005) was used toanalyze the significance difference. The correlation coefficients fordifferent variables were tested using the Spearman rank ordercorrelation test (Richard Lowry, 1998, 2008). Pvalues less than 0.05(two-tailed) were considered significant.

B. Arsenic metabolites in mice tissues after administering a single intra-muscular dose of sodium arsenate ³⁴ .

Reagents³⁴. TRIZMA (Reagent grade, minimum 99.95), bovine serum albumin, and glutathione (GSH) were purchased from Sigma Chemical Co.(St. Louis, MO). [¹⁴C]-MMA(V) (0.55 μCi/nmol, 98% purity) was synthesized by Professor Eugene A. Mash, Jr., Department of Chemistry, The University of Arizona. Disodium methylarsenate wasobtained from ChemService, Inc. (WestChester, PA). Monoflow-3 scintillation cocktail was from National Diagnostics(Atlanta, GA). Sodium arsenate (ACS reagent grade) was purchased from MCBReagents (Cincinnati, OH). Diethyldithiocarbamic acid diethylammonium salt(DDDC) and carbon tetrachloride (CCl₄, 99.5+% anhydrous) were analyticalreagent grade and were purchased from Aldrich Chemical Co., Inc. All otherchemicals were analytical reagent grade or of the highest quality obtainable.Water was doubly deionized and distilled.

Animals³⁴. Male wild type (WT) mice (DBA/1LacJ), 8 weeks of age, were purchased from Jackson Laboratory, Bar Harbor, Maine, USA. Upon arrival, mice were acclimated for at least 1 week and maintained in an environmentally controlled animal facility operating on a 12-h dark/12-h light cycle and 22–24°C. They were provided with Teklad (Indianapolis, IN) NIH-31 Modified Mouse/Rat Sterilizable Diet #7013 and water, ad libitum.

Preparation of the homogenate for analysis of arsenic species³⁴. Wildtype (WTmale, average age 10.3 weeks, n=18) mice were administered a single intra-muscular dose of sodium arsenate (4.16 mg As/kg body weight) inside of the left hind leg. In order to observe the metabolites of arsenate within a short period of time after a single injection of arsenate, we chose im injection because this was a consistent and less error prone way to get the arsenate into the body. We wished to detect MMA(III) and DMA (III) as quickly as possible before they were further metabolized. At the following times, 0 (control), 0.5, 1, 2, 4, 8, and 12 h, mice were decapitated rapidly by guillotine. Liver, kidneys, urinary bladder tissue, lungs, testes, and heart were removed, blotted on tissue papers (Kimtech Science, Precision Wipes), and weighed. The mouse tissues were homogenized in 0.02 M Tris–HCl (pH 8.0) and 20 mM DDDC at 4°C (cold room),using a glass homogenizer and Teflon coated steel pestle, transferred to a 10-mL acid-washed polypropylene tube and extracted immediately. The samples were always kept on dry ice. Protein concentration was determined by the method of Bradford³⁶, using bovine serum albumin as a standard.

Extraction of arsenic species³⁴. Arsenic species were extracted from homogenates with CCl₄ and 20 mM DDDC by continuous shaking for 30 min at room temperature (Fig. 3) as described previously³⁵. After centrifugation (11,000×g for 15 min at 4°C), an organic phase, aqueous phase, and interphase were obtained and treated as follows:

The organic phase (trivalent species)³⁴ was transferred to another tube and back extracted two times into 200 μL of 0.1MNaOHby shaking for 30 min at room temperature; centrifuged (11,000×g at 4°C) for 15 min; and the NaOH phase (upper phase) transferred to another 5 mL acid-washed polypropylene tube.

The aqueous phase (pentavalent species)³⁴ was transferred to another acid-washed polypropylene tube.

The inter-phase (trivalent and pentavalent species)³⁴ was homogenized in 0.020 M Tris–HCl (pH 8.0) and 20 mM DDDC and extracted twice with CCl₄ and 20 mM DDDC. The NaOH phase, containing +3 oxidation state arsenicals, and the aqueous phase, containing +5 oxidation state arsenicals, were each oxidized by addition of H₂O₂ to a final concentration of 5%, mixed and incubated over night at room temperature. The following morning, each sample (aqueous or NaOH phase) was boiled for 10 min. The aqueous phase was centrifuged (11,000×g for 15 min, 4°C). Each supernatant of NaOH phase and aqueous phase was transferred to separate acid-washed Eppendorf tubes. The pH was adjusted (5.5 to 6.5) by using NaOH or HCl, and the tubes were centrifuged again (14,000×g for 10 min at 4°C). Finally, the supernatant was transferred to another Eppendorf tube and stored at −20°C for HPLC-ICP-MS analysis. Fig. 3 shows the diagram of this procedure.

HPLC-ICP-MS assay for arsenic species³⁴. The HPLC system consisted of an Agilent 1100 HPLC (Agilent Technologies, Inc.) with a reverse-phase C18 column [Prodigy 3μ ODS, 150×4.60 mm, Phenomenex, Torrance, CA]. The mobile phase (pH 5.85) contained 4.7 mM tetrabutylammonium hydroxide, 2 mM malonic acid, and 4% (v/v) methanol at a flow rate of 1.2 mL/min. The column temperature was maintained at 50°C. This was essentially the method of Gong et al. (2001)³¹, except ICP-MS instead of HG-AFS was used. An Agilent 7500a ICP-MS with a Babington nebulizer was used as the detector. The operating parameters were as follows: R_f power 1500 W, plasma gas flow 15 L/min, carrier flow 1.2 L/min, and arsenic was measured at 75 m/z. The validity of the procedure used for analysis has been published previously^34,35.

Results and Discussion

Arsenic metabolites including MMA^III and DMA^III in human urine. Urine and blood samples were collected from 75 subjects in theLagunera area of Mexico. There were four groups, based on total arsenic concentrations in their drinking water(9-100μg/L).After collection, the urine samples were immediately frozen in a portable icebox, containing dry ice.The samples were kept frozen while being transported to the University of Arizona, Tucson where theywerestored at -70°C before analysis.

Six different arsenic metabolites including MMA(III) and DMA(III) were measured in urines of four groups, based on total arsenic concentrations in their drinking water(9-100μg/L)from people (n=75) of Mexico. After acid digestion, total arsenic wasalso measured on the same urine samples by using ICP-MS, and the results show that the average concentration of total arsenic in urines (µg/L) of the corresponding groups were very close with the average sum of arsenic metabolites (µg/L)of the same urine samples (Table 1). These results showvery little or no AsB (BDL)in these urine samples.The results also indicate that the people in our study may have drunk water from different sources because arsenic in drinking water were not strongly correlated with the total arsenic in their urines.

The detection and quantification of urinary MMA(III) andDMA(III) in various populations have been reported over the past20 years but with highly variable findings. Some reports suggest that in exposed populations, these trivalent compounds are major or even dominant components of urinary arsenic^18,19,27. Other investigators suggest little or no DMA(III), and very low levels ofMMA(III) occur in populations with a range of arsenicexposures^28-30.In our study, we could not detect DMA(III), and very low levels of MMA(III) in a few urine samples occurred in populations of Mexico with a range of arsenic exposure from 9-100 µg/L (Table 2).Detectable MMA(III) was found in a total of 14(~18%) out of 75 cases. Kalman, et.al. (2014)³⁷ was reported that they found detectable MMA(III) in a total of 41(~6%) of 643 cases.

The average concentration of MMA(III) in human urine of arsenic exposed people in the Lagunera area of Mexico were0.17µg/L(n=6), 0.08µg/L(n=2), and 0.12µg/L(n=7) out of total 19 (group 4), 19 (group 1), and 17 (group 2) urine samples, respectively (Table 2). MMA(III) was not detectable in urines of group 3 people(n=20),but most of the metabolite was DMA(V) (~64% of total urinary As),where total As concentration in their drinking water was 52 µg/L. This meansMMA(V) was reduced to MMA(III);then, MMA(III) methylated to DMA(V), even though there was no detectable MMA(III) in urines of group 3 people. There was also important informationfromthese study groups thatDMA(III) was not detectable in any of these urine samples (n=75).These results indicate that DMA(III) could be more instable comparedto MMA(III),and reports also suggest that these trivalent compounds, MMA(III) and DMA(III),would not be the dominant components in urinary arsenic of this arsenic exposed people in Mexico.

Questions: Why was MMA(III) found in a few urine samples (n=14) out of total 75 samples? Why was DMA(III) not found/detected in any of these urine samples (n=75)? Does it oxidize during analyses? Does it depend on arsenic concentration in drinking water? Does it depend on the physiological/biological/environmental conditions of humans? Does it depend on the storage condition and duration of storage time? Does it depend on the genetic variability of humans? Does it depend on the reductase activities of humans? Does it oxidize before urine collection? What else?

ND, not detectable

The average percentage of MMA(III) in urine samples of arsenic exposed people in the Lagunera area of Mexico were 0.44%, 0.26%, N.D. (Not Detectable), and 0.20% for the groups 4, 1, 3, and 2 where arsenic concentrations in their drinking water were 9, 17.5, 52, and 100 µg/L, respectively (Table 3). Thepercentage of MMA(III)was at very low levels compared with other arsenic metabolites detected or measured in these urine samples. The highest percentage of arsenic metabolite was DMA(V)(61% to 74% of the total arsenic in urine after acid digestion),and it indicates that most of the MMA(III) converted to DMA(V)↔ DMA(III), but DMA(III) may be very instable and oxidized to DMA(V) very quickly.

ND, not detectable

Reductase activities in human blood

As(V) Reductase Assay. The reaction mixture (100 µL), containing 0.1 M MOPS buffer, pH 7.5, 1.0 mM EDTA, 10 mM inosine, 0.37-6 mM arsenate, 0.5 µCi of carrier-free [⁷³As]arsenate, 0.5-5 mM DHLP, and human blood arsenate reductase, was incubated for 30 min at 37°C.Arsenite, 0.1 M (100 µL) after incubation, was added rapidly, and the tubes were immersed in a boiling water bath for 2 min, cooled on ice for 5 min, and then centrifuged at 12,000rpm for 6 min. A 100 µL sample of the supernatant was injected into a 4.9 mm x 250 mm Hamilton PRP-X100 anion exchange column, using 30 mM sodium phosphate (pH 5.0), with a flowrate of 1.45 mL min^-1. The arsenite and arsenate were quantified, using a post column inline Beckman 171 radioisotope detector. Monoflow III was used as the scintillation fluid, anda flow rate was used, and it was 3 times that of the mobile phase (Radabaugh et al. 2002)³⁸.

MMA(V) Reductase Assay. The reaction mixture contained 0.1M Tris HCL (pH 8.0), 5 mM GSH, 15 mM MMA(V), 8.1 x 109 cpm of [¹⁴C]MMA(V), and human blood in a final volume of 250 μL and incubated for 60 min at 37°C. The MMA(III) that was produced was isolated by the extraction method previously described^34,39,40,41.

DMA(V) Reductase Assay. The reaction mixture (250 μL) was the same as in MMA(V) reductase assay, except that 8.2 x 109 cpm of [¹⁴C]DMA(V), and 10 mM sodium DMA(V) replaced MMA(V). The product of the reaction that was formed was measured as previously described^34,39,40,41.

Protein Assay. Protein concentrations were determined using bovine serum albumin as the standard⁴².

(a) As(V) reductase activities

Human blood samples were collected from 75 subjects in theLagunera area of Mexico. There were four groups, based on totalarsenic concentrationsin their drinking water(9-100μg/L).Blood was collected by venous puncture, intoVacutainers containing EDTA, transferred to the vial, and immediately frozen. The samples were kept frozen while being transported to the University of Arizona, Tucson where theywerestored at -70°C before analysis.

The average As(V) reductase activities in bloods of our study population were 122.02±3.96,115.44±4.51, 118.21±4.00, and 120.19±6.39As(III) formed/ mg protein/hfor groups 4, 1, 3, and 2, respectively (Table 4).The results show that As(V) reductase activities in bloods of different groups of humanswere very similar, and these reductase activities were not dependent on the arsenic concentrations in their drinking water or urinary arsenic.

(b) MMA(V) and DMA(V) reductase activities ^41,43

Blood samples were collected from different exposure groups of 191 subjects (98 females and 93 males), aged 18-77 years in the Lagunera area of Mexico⁴³. There were five groups, based on total arsenic concentrations (38-116 μg/L) in their drinking water (Table 5). The general characteristics of the study population have been previously described in detail⁴³. Human blood samples were collected and immediately frozen. The samples were kept frozen while being transported to the University of Arizona, Tucson where theywerestored at -70°C before analysis.

The activities of MMA(V) reductase were significantly lower than DMA(V) reductase activities in the bloods of our study population (p<0.01; Mean values: 3.87±0.22 MMA(III) formed/ mg protein/h versus 6.34±0.22 DMA(III) formed/ mg protein/h, respectively). These reductase activities in blood between females and males of different arsenic exposure groups were almost similar, except for a few cases (Fig. 5). The results indicate that the activities of MMA(V) reductase and DMA(V) reductase in humans’blooddon’t depend on the arsenic level in their urinary arsenic/arsenic concentration in their drinking water (Figs. 4 and 5).

The correlations between MMA(V) reductase and DMA(V) reductase activities in blood of both females and males were very weak (r= +0.06 and r= -0.03, respectively). These correlations data indicate that there maybe two different enzymes, and the mechanisms of reduction activities could be different. The activities of MMA(V) reductase and DMA(V) reductase were not depended on ages of these population (r= -0.05 and r= -0.11, respectively) too. A weak correlation found between MMA(V) reductase activities in blood and % MMA(V) in urine of males (r= -0.14, p=0.19) but not of females (r= -0.01, p=0.93). We also found a negative and statistically significant correlation between DMA(V) reductase activities in blood and %DMA(V) in urine of males (r= -0.23, p<0.05). This correlation was not statistically significant for females (r=-0.12, p=0.25).

Distribution of MMA ^III and DMA ^III in animal tissues ³⁴

The chemical forms of arsenic determine the toxicity and bioavailability of arsenic compounds^7,15. MMA(III) and DMA(III) are more potent cytotoxins,genotoxins, and enzyme inhibitors than are inorganic arsenate,arsenite, or the +5-oxidation state methylated arsenicmetabolites, MMA(V) and DMA(V)^20-25.

When mice were injected intramuscularly with Na arsenate (4.16 mg As/kg body weight); tissues removed at 0.5, 1, 2, 4, 8, and 12 h after arsenate injection; and the arsenic species measured by HPLC-ICP-MS. The results indicated that the highest concentration of the very toxic MMA(III), a key biotransformation, was in the kidneys, but DMA(III) was in the urinary bladder tissue of mice(Fig. 6)³⁴.

The distribution of arsenic metabolites, including the very toxic MMA(III) and DMA(III) in mice tissues, will be emphasized in the present discussion, andthese resultswould help to know their distribution in urines. Sampayo-Reyes et al. (2000)³⁵ detected MMA(III) and DMA (III) in hamster liver 15 h after a single ip dose of arsenate. MMA(III) has also been reported in rat liver 2 h after iv injection of arsenate⁴⁴, although the use of the rat as a useful model for the human is of questionable and limited value².

The concentration of the very toxic MMA(III) was significantly higher than that of MMA(V) in all of the tissues tested (Liver, kidneys, urinary bladder tissue, lungs, testes, and heart). This is a validation of the extraction procedure used to measure the relatively unstable MMA(III).At 2 h after injection, the highest concentration of MMA(III) was in the kidneys, which was greater than the urinary bladder tissue, testes, heart (p<0.001), or liver and lungs (p<0.01) of mice³⁴.Compared with the other species of arsenic, MMA(V)concentrations were the lowest in all tissues examined³⁴.

On the other hand, the concentration of DMA(V) was higher than DMA(III) in the liver,kidneys,urinary bladder tissue, lungs, and testes at all times. DMA(V) accumulated at a higher concentration in the lungs and urinary bladder tissue, but the concentration of DMA(III) was highest in the urinary bladder tissue, followed by the kidneys, lungs, heart, testes, and liver.In all of the tissues, both DMA(III) and DMA(V) were the highest at 4 h for the WT mice. The concentration of DMA(III) was significantly higher in the urinary bladder tissue than in other tissues (p<0.05), except in the kidneys of the mice.

According to these results, the concentration of MMA(III) was significantly higher than that of MMA(V) in all of the tissues tested (Liver, kidneys, urinary bladder tissue, lungs, testes, and heart). But the concentration of DMA(V) was higher than DMA(III) in the liver,kidneys,urinary bladder tissue, lungs, and testes at all times. The results indicate that MMA(V) reduced to MMA(III) faster compared to DMA(V) to DMA(III) because we found significantly high amounts of MMA(III) in all the tissues, compared to MMA(V) at the same time (2 h), but DMA(V) was significantly high in the tissues, compared to DMA(III) at 4 h. It could be all or most of the MMA(V) reduced to MMA(III) and methylated to DMA(V), but less percentage of this DMA(V) reduced to DMA(III). It might bedue to the fact that DMA(III) was more instable in the tissues,compared to MMA(III) and oxidized faster, compared to MMA(III). These results also indicate that due to the instability and faster oxidation of DMA(III), we could not measure/detect it, but we found low percentage of MMA(III) in urines of some arsenic exposed people due toits more stabilityand bonding capacitywith tissues, comparing to DMA(III). May be due to genetic variability and other factors, we found MMA(III) in urines of some of them but not allin our study groups.

MMA(V) reducing activity of WT mice tissues ³⁴

The rate of MMA(V) reducing activity was determined in various tissues, including blood hemolysates of WT mice. The indicated results are based on the rate (limiting amounts) and not the limit (enzyme excess). For the wild-type mice, the urinary bladder tissue had the largest enzyme activity, and the bloodhemolysates had the smallest³⁴. The order of MMA(V) reductase activity to highest to lowest are bladder, heart, brain, lungs, spleen, skin, kidneys, liver, testes, and blood hemolysates. The mechanism of inorganic arsenic biotransformation and the stability or instability of more toxic MMA(III) and DMA(III) maydepend on biological environment and genetic variability, but more experiments are needed to understand these mechanisms.

Are MMA(III) and DMA(III) oxidized before urine collection ?

There was no detectable levels MMA(III),but it was 64% DMA(V) in urines of group 3 people (n=20) (Table 3). That means, MMA(V) was reduced to MMA(III) and then MMA(III) methylated to DMA(V), even though there was no detectable MMA(III) in urines of this group 3 people. Another important information of this study group is that DMA(III) was not measured in any of these urine samples (n=75). These results indicate that DMA(III) could be more instable and/or produced very less amount, comparing with MMA(III) and suggest that the +5-oxidation state of arsenic metabolite, DMA(V), could be the most dominant methylated arsenic metabolite in humans’ urine of this arsenic exposed people.

The average percentages of MMA(III) were 0.44%, 0.26%, N.D. (Not Detectable), and 0.20%,but the percentages of DMA(V) were 74.43%, 62.23%, 64.38%, and 60.78%in urines for the population of the group’s 4, 1, 3, and 2, where arsenic concentrations in their drinking water were 9, 17.5, 52, and 100 (µg/L), respectively. There was a small percentage of MMA(III), compared with other arsenic metabolites, in these urine samples, but there was no detectable DMA(III). The highest percentage of arsenic metabolite was DMA(V) (61% to 74%) of the total arsenic in urines, and it indicates that most of the MMA(III) converted to DMA(V) ↔ DMA(III), but DMA(III) may be very instable and oxidized to DMA(V), quickly.

In animal experiment, mice were injected intramuscularly with Na arsenate and the arsenic species in tissues measured by HPLC-ICP-MS. The results indicated that the highest concentration of very toxic MMA(III), a key biotransformant was in the kidneys, but DMA(III) was in the urinary bladder tissue of mice³⁴.

The concentration of the very toxic MMA(III) was significantly higher than that of MMA(V) in all of the tissues tested at all times after arsenate injection. At 2 h after injection, the highest concentration of MMA(III) was in the kidneys, which was greater than the urinary bladder tissue, testes, heart (p<0.001), or liver and lungs (p<0.01) of mice. Compared with the other metabolites of arsenic, MMA(V)concentrations were the lowest in all tissues examined³⁴, i.e., most of the MMA(V) were reduced to MMA(III)immediately and then it was methylated to DMA(V). However, some percentage of MMA(III) could also be oxidized to MMA(V) before methylated to DMA(V).

On the other hand, the concentration of DMA(V) was higher than DMA(III) in the liver,kidneys,urinary bladder tissue, lungs, and testes at all times. DMA(V) accumulated at a higher concentration in the urinary bladder tissue and lungs, but the concentration of DMA(III) was highest in the urinary bladder tissue followed by the kidneys, lungs, heart, testes, and liver. In all of the tissues, both DMA(III) and DMA(V) were highest at 4 h for the WT mice. The concentration of DMA(III) was significantly higher in the urinary bladder tissue than in other tissues (p<0.05), except in the kidneys of the mice.The results indicate that maybe the less percentage of DMA(V) was reduced to DMA(III) or after reduction it may be immediately oxidized to DMA(V) in tissues.

The highest concentration of As(V) was in the urinary bladder tissue (~30 µg/gm tissue) and the kidneys (~11 µg/gm tissue)at 0.5h³⁴.But the concentration of As(III) and MMA(III) were highest in kidneys,compared to bladder tissue. Specially, MMA(III) was ~2 µg/gm tissue vs ~0.3 µg/gm tissue in kidneys vs. bladder tissues at the same time (2h), respectively. Even though MMA(III) was significantly low in bladder tissues compared to kidneys, the highest concentration of both DMA(V) and DMA(III) were measured in bladder tissues compared with all of the tissues tested(for example, ~2.2µg/gm tissue vs ~1.4 µg/gm tissue of DMA(V) and ~1µg/gm tissue vs ~0.6µg/gm tissue of DMA(III) for bladder tissue vs kidneys at 4h, respectively).The results indicate that it could be DMA(V) accumulated in the bladder, and DMA(V) would be the most dominated arsenic metabolite in the bladder tissue fluid.

According to the results, the concentration of MMA(III) was significantly higher than that of MMA(V) in all of the tissues tested (Liver, kidneys, urinary bladder tissue, lungs, testes, and heart).On the other hand, the concentration of DMA(V) was higher than DMA(III) in the liver,kidneys,urinary bladder tissue, lungs, and testes at all times. The results indicate that MMA(V) reduced to MMA(III) faster compared to DMA(V) to DMA(III) because we found significantly highest amount of MMA(III) in all the tissues, compared to MMA(V) at the same time (2 h), but DMA(V) was significantly highest in the tissues, compared to DMA(III) at 4 h. It could be that most of the MMA(V) reduced to MMA(III) and methylated to DMA(V), but less or more percentage of this DMA(V) reduced to DMA(III), but DMA(III) may be very instable and oxidized to DMA(V), quickly and found higher percentage of DMA(V) compared to DMA(III) in tissues. It might be because DMA(III) was more instable in the tissues,comparedto MMA(III), and oxidized faster, compared to MMA(III). It could also be suitable for binding of MMA(III) to tissue protein components,compared to DMA(III).

In our study groups, we could not detect DMA(III), and low levels of MMA(III) in a few urine samples occurred in populations of Mexico with a range of arsenic concentration in their drinking water from 9-100 µg/L. We may explain the distribution of MMA(III) and DMA(III)in urines of humans from the distribution of MMA(III) and DMA(III) in tissues of mice because DMA(III) could be more instable in the tissues and oxidized faster, compared to MMA(III). Therefore, due to the instability and faster oxidation of DMA(III), we could not detect DMA(III), but we found some percentage of MMA(III) in some urine samples of arsenic exposed people due to its more stability, compared to DMA(III). In tissues, the concentration of DMA(V) was higher than DMA(III) in the liver,kidneys,urinary bladder tissue, lungs, and testes at all times. Most of the MMA(III) was methylated to DMA(V), and DMA(V) was the dominated metabolite in tissues,startingat 4h,which wasalso true for human urine because DMA(V) was the dominated arsenic metabolite in there.We detected both MMA(III) and DMA(III) in tissues but not in urines, and the reports suggested that tissue levels of MMA(III) and DMA(III)are both more relevant and less susceptible to oxidative artifacts than are urine samples.

It has been reported that inorganic arsenic is commonly methylated in liver,^45,46 but in our mouse studies we found that the highest amount of MMA(III) was in kidney, followed by liver and bladder. On the other hand, both DMA(V) and DMA(III) were highest in bladder followed by kidney>liver³⁴. The concentrations of MMA(V) were higher in kidney and bladder tissues than liver,and MMA(V)concentrations were the lowest,comparing with other arsenic metabolites in all tissues examined³⁴.If liver is the main/common organ for arsenic methylation,then liver should contain more MMA(III) and DMA(III), but the results show different. These results indicate that maybe MMA(III) and DMA(III) bonding proteins/components in liver maybe less or weak,comparing to bladder, kidneys, etc., and migratedthese toxic metabolites to other tissues, or the liver may not be the only most common organ of inorganic methylation/biotransformation process in vivo.

The urine flows from the kidneys through the ureters to the bladder. The urine is stored in the bladder until it leaves the bladder through the urethra. Urine is typically the same as a person's body temperature. On average, this is 98.6^oF (37^oC). Some people have normal temperature variations that may be slightly hotter or slightly cooler than this. Urine will usually maintain its temperature outside the body for about four minutes. If MMA(III) and DMA(III) metabolites could survive at body temperature (37^oC),then why aren’t they stable at frozen or below frozen temperature(-20^oC, -70^oC, etc)?Also, the concentration of MMA(III) does not depend on the concentrations of total arsenic in urines. May be, most of them are oxidized before urine collection, and a small percentage of MMA(III) and DMA(III) are bound with some components/proteins in urine under biological environment, which are not suitable under invitro conditions, even though they are kept at frozen or below frozen.

Stability reportsof MMA(III) and DMA(III) from other researchers

Potential problems in the determination of MMA(III) that have been noted in the past include storage instability and measurement artifacts^47,48. Based on the reported storage instability of MMA(III) in urine samples at –20^oC, negative findings for several of these studies^49-54 might be due to oxidation during storage, while findings of high levels and high proportions of MMA(III) suggest other method artifacts. Laboratory studies ofMMA(III) instability have led to conclusions that it might be more prevalent than reported in urine samples from population studies, due to losses in sample handling and measurement⁵⁵. Lindberg and co-workers⁴⁹ have suggested that MMA(III) may be too reactive toward oxidation or binding to tissue components to be suitable for use as a urinary biomarker. More recent reports have suggested that tissue levels are both more relevant and less susceptible to oxidative artifacts than urine samples⁵⁶.

The wide variation in reports of the frequency, proportion, andlevel of MMA(III) and DMA(III) in human urine may indicate variability due to a variety of factors, summarized recently byTseng⁵⁷. These include sample collection, preservation, and measurement; population-specific environmental or nutritional factors; or underlying genetic differences^58-60, related to the enzymes that control biotransformation. Resolving the questions regarding issues of sample collection/storage/analysis is therefore of prime importance in determining the biological significance of measured levels of trivalent methylated arsenic in urine.

Varying degrees of instability of MMA(III) in solution, and the role of sample matrix, temperature, and pH have been noted previously. Arsenic species (excluding trivalent methylated forms)in aqueous samples are reported to be less stable than either fresh or reconstituted freeze-dried urine⁶¹, but several reports indicate that MMA(III) is more unstable in urine than in water^47,53,62. Of equal significance, variable stability in different urine samples has been noted⁶². Using MMA(III)-fortified urine samples, Gong et al.⁴⁷found 30% loss after 1 day, at both 4^oC and –20^oC. The losses ofMMA(III) from urine over 1 to 3 days at 25^oC, reported by Gong and his co-workers⁴⁷, are particularly significant because that is the time scale and temperature for samples during the actual analysis.

Conclusion

Analysis of urinary arsenic species in samples (n=75), having total urinary arsenic of30 to 59 µg/L, showed few cases (~18%) of detectable MMA(III). The finding from this study of mostly undetectable MMA(III), and there were nodetectable levels of DMA(III)in these urine samples. The highest percentage of arsenic metabolite was DMA(V) (61% to 74%) of the total arsenic, and it indicates that most of the MMA(III) converted to DMA(V) and a part of this DMA(V) ↔ DMA(III), but it may be very unstable and oxidized to DMA(V) again, quickly. It seems that maybe both MMA(III) and DMA(III) are unstable, but DMA(III) could be more unstable comparing MMA(III).

The results indicate that MMA(V) reduced to MMA(III) faster, compared to DMA(V) to DMA(III), because there was a significantly high amount of MMA(III) in all the tissues, compared to MMA(V) at the same time (2 h), but DMA(V) was higher in most of the tissues, compared to DMA(III), and it was highest at 4 h. The concentration of the very toxic MMA(III) was significantly higher than that of MMA(V) in all of the tissues tested (Liver, kidneys, urinary bladder tissue, lungs, testes, and heart) at 0.5, 1, 2, 4, 8, and 12h after arsenate injection. On the other hand, the concentration of DMA(V) was higher than DMA(III) in the liver,kidneys,urinary bladder tissue, lungs, and testes at all times.It could be all or most of the MMA(V) reduced to MMA(III) and methylated to DMA(V) but less percentage of DMA(V) reduced to DMA(III). Maybe due to the fact that DMA(III) was more unstable in the tissues comparing MMA(III) and oxidized faster compared to MMA(III). These results also indicate that due to the instability and faster oxidation of DMA(III) we could not measure/detect DMA(III), but due to comparing more stability of MMA(III), we found low levels of MMA(III) in urine of exposed people.

Finally, the trivalent MMA(III) compound could be the most dominant toxic arsenic metabolite in humans’ tissues for a period oftime but not in urine because arsenic metabolism/biotransformation process occurred in tissues, and urine contains the end products only. On the other hand, DMA(V),instead of DMA(III),would be another dominant arsenic metabolite in tissues as well as in urine. Their stability/instability may depend on biological environment, genetic variability, and other complex factors. More experiments are needed to understand the mechanism of inorganic arsenic biotransformation, and stability or instability of very toxic MMA(III) and DMA(III).

Acknowledgement

The Author wants to dedicate this paper to the memory of Dr. H. Vasken Aposhian and Mary M. Aposhian who passed away on September 6, 2019, and September 9, 2009, respectively. Prof. VasAposhian and MaryAposhian had collected these urine and blood samples from people in the Lagunera area of Mexico. Dr. Aposhian was anEmeritus Professor in the Department of Molecular and Cellular Biology at the University of Arizona, and he was the author’s supervisor.

The Author would like to thank Ms. Shreya Chowdhury for reading, reviewing, and editing this manuscript.

The Superfund Basic Research Program NIEHS Grant Number ES 04940 from the National Institute of Environmental Health Sciences and the Southwest Environmental Health Sciences Center P30-ES-06694 supported this work.

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