Tissue Distribution and Urinary Excretion of Inorganic Arsenic and Its Methylated Metabolites in Mice Following Acute Oral Administration of Arsenate

E. M. Kenyon*,1, L. M. Del Razo{dagger} and M. F. Hughes*,{dagger}

* Pharmacokinetics Branch, Experimental Toxicology Division, Mail Stop B143–01, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, and {dagger} Toxicology Department, CINVESTAV-IPN, Mexico City, Mexico

1 To whom correspondence should be addressed. Fax: (919) 541-5394. E-mail: kenyon.elaina{at}epa.gov.

Received December 7, 2004; accepted February 2, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The relationship of exposure dose and tissue concentration of parent chemical and metabolites is a critical issue in cases where toxicity may be mediated by a metabolite or by parent chemical and metabolite acting together. This has emerged as an issue for inorganic arsenic (iAs), because both its trivalent and pentavalent methylated metabolites have unique toxicities; the methylated trivalent metabolites also exhibit greater potency than trivalent inorganic arsenic (arsenite, AsIII) for some endpoints. In this study, the time-course tissue distributions for iAs and its methylated metabolites were determined in blood, liver, lung, and kidney of female B6C3F1 mice given a single oral dose of 0, 10, or 100 µmol As/kg (sodium arsenate, AsV). Compared to other organs, blood concentrations of iAs, mono- (MMA), and dimethylated arsenic (DMA) were uniformly lower across both dose levels and time points. Liver and kidney concentrations of iAs were similar at both dose levels and peaked at 1 h post dosing. Inorganic As was the predominant arsenical in liver and kidney up to 1 and 2 h post dosing, with 10 and 100 µmol As/kg, respectively. At later times, DMA was the predominant metabolite in liver and kidney. By 1 h post dosing, concentrations of MMA in kidney were 3- to 4-fold higher compared to other tissues. Peak concentrations of DMA in kidney were achieved at 2 h post dosing for both dose levels. Notably, DMA was the predominant metabolite in lung at all time points following dosing with 10 µmol As/kg. DMA concentration in lung equaled or exceeded that of other tissues from 4 h post dosing onward for both dose levels. These data demonstrate distinct organ-specific differences in the distribution and methylation of iAs and its methylated metabolites after exposure to AsV that should be considered when investigating mechanisms of arsenic-induced toxicity and carcinogenicity.

Key Words: inorganic arsenic; metabolites; dimethylated arsenic; monomethylated arsenic.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The metalloid inorganic arsenic (iAs) is found naturally at varying levels in water, soil, air, and food. The major public health concern for iAs is the association of exposure, primarily via drinking water, with development of cancers of the skin and internal organs (National Research Council, 1999Go, 2001Go). The citizens of several countries, including India and Bangladesh, are facing a public health crisis from their use of groundwater, contaminated with high levels of iAs, as their source of drinking water (Alam et al., 2002Go; McLellan, 2002Go; Rahman et al., 2001Go). In the United States, the potential development of cancer from exposure to iAs-contaminated drinking water recently led to the lowering of the iAs drinking water standard from 50 ppb to 10 ppb (U.S. EPA, 2001Go).

An intriguing problem with iAs and cancer is identification of the active agent(s). From the form of iAs in drinking water, and its resultant metabolism, an individual may be exposed to at least six different arsenicals. Inorganic arsenic is generally found in drinking water as either arsenate (AsV) or arsenite (AsIII). Arsenate is found primarily in oxygenated waters, whereas arsenite is detected in more reducing environments. Once arsenate enters the gastrointestinal tract or is absorbed, it is rapidly reduced to arsenite. In vitro studies have shown that arsenate reduction can occur nonenzymatically in the presence of thiols in a reducing (low oxygen) environment, but more recent in vitro evidence suggests the reduction also occurs enzymatically (Thomas et al., 2001Go, 2004Go). A purine nucleoside phosphorylase catalyzes the in vitro reduction of arsenate (Gregus and Nemeti, 2002Go; Radabaugh et al., 2002Go), but its in vivo relevance is not clear (Nemeti et al., 2003Go; Nemeti and Gregus, 2004Go). Arsenite is oxidatively methylated to monomethylarsonic acid (MMAV) by an arsenic methyltransferase. MMAV is then reduced to monomethylarsonous acid (MMAIII), which is then methylated to dimethylarsinic acid (DMAV). DMAV may also be subsequently reduced to dimethylarsinous acid (DMAIII). An enzyme, AS3MT (E.C.2.1.1.138), has been isolated from rat liver that catalyzes both the oxidative methylation and reduction of arsenic (Lin et al., 2002Go; Waters et al., 2004Go).

The analysis of urine collected from humans and laboratory animals exposed to iAs has been the primary means of studying iAs metabolism. Arsenic is excreted primarily in urine by most mammalian species, with DMAV being the main product eliminated, after exposure to iAs. In the past, As methylation had been thought to be solely a detoxication reaction, because it facilitates excretion of arsenic, and the pentavalent methylated forms of arsenic (MMAV and DMAV) commonly detected in urine are relatively nontoxic following acute exposure compared to either AsV or AsIII. However, there has been growing recognition that methylation is actually an activating process, because MMAIII and DMAIII are potent toxicants, even more toxic than arsenite for certain DNA-damage endpoints (Ahmad et al., 2002Go; Mass et al., 2001Go). Several studies have also demonstrated that DMAV is a bladder carcinogen and tumor promoter in rodents, albeit at relatively high doses (see reviews by Kenyon and Hughes, 2001Go; Hughes, 2002Go; Wanibuchi et al., 2004Go). Cohen et al. (2002)Go have presented evidence to suggest that the DMAIII formed in vivo from DMAV is the active agent in bladder cell proliferation and cytotoxicity observed in rats exposed to DMAV. Recent studies have detected both MMAIII and DMAIII in urine of humans exposed to iAs (Aposhian et al., 2000Go; Del Razo et al., 2001Go; Le et al., 2000Go; Mandal et al., 2001Go; Valenzuela et al., in press), suggesting that these toxic metabolites are formed in vivo and have the potential for transport to target tissues. The actual identity of DMAIII in urine has been questioned by Hansen et al. (2004)Go. They suggest that the DMAIII in humans might actually be a sulfur-containing arsenical. However, Hansen et al. (2004)Go collected and analyzed urine from sheep, a ruminant, fed seaweed that contains arsenosugars, from which the dimethylarsinic metabolites are derived.

The use of urinary metabolite data collected from arsenic-exposed humans and rodent models as a surrogate for arsenic tissue dosimetry is a significant uncertainty in risk assessment because it may not adequately reflect target tissue distribution. In a recent study in the mouse, Hughes et al. (2003)Go reported that after 10 days of once-daily exposure to arsenate (0.5 mg As/kg) by oral gavage, the distribution of urinary arsenicals was: DMA(III+V), approximately 95%; iAs(III+V), 5% or less, and MMA(III+V), less than 1%. However the tissue distribution in similarly exposed mice was quite different. MMA(III+V) was detected in liver, blood, lung, and kidney with a distribution from 3–44%, but not the urinary bladder. The iAs(III+V) distribution ranged from 12 to 57%, and that of DMA(III+V) ranged from 35–88%. After repeated exposure, the tissue with the highest distribution of MMA(III+V) was blood (44%), DMA(III+V) was bladder (88%), and iAs(III+V) was kidney (57%). Thus, measurement of metabolites in urine may not give a clear indication of the true tissue distribution of speciated arsenic.

The objective of this study was to examine the relationship of acute exposure to iAs (as arsenate) with the tissue concentrations of iAs and methylated metabolites. This is a critical issue for risk assessment, because iAs and its methylated metabolites have unique toxicological potentials. The deleterious effects of arsenic may also be due to the action of multiple arsenicals at different stages in the process of eliciting a toxic or carcinogenic response.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Sodium arsenite and sodium arsenate were obtained from Sigma Chemical Co. (St. Louis, MO). MMAV and DMAV were obtained from Ventron (Danvers, MA) and Nakarai Chem (Kyoto, Japan), respectively. Sodium borohydride was obtained from EM Science (Gibbstown, NJ). Hydrochloric acid (HCl) was obtained from Fisher Scientific Co (Fairlawn, NJ). Dried Bovine Liver Standard Reference Material (SRM1577b) for toxic metals was obtained from the U.S. National Institute of Standards and Technology (Gaithersburg, MD). All other chemicals used were of the highest grade commercially available.

Animals.
Ninety-day-old female B6C3F1 mice were obtained from Charles River Laboratories (Raleigh, NC) and held for 2 weeks prior to commencing studies. The animals were maintained according to the guidelines in the NIH Guide to the Care and Use of Laboratory Animals within an AAALAC-approved animal facility. They were housed in polycarbonate shoebox cages (10–11/cage) with hardwood chip bedding and were provided with Rodent Chow (Purina, St. Louis, MO) containing <1 ppm arsenic and tap water ad libitum. The room was kept on a 12/12-h light/dark cycle and at a temperature of 22 ± 1°C and humidity of 50 ± 10%.

Treatment.
Sodium arsenate was dissolved in HPLC grade water and administered as a single oral dose at 10, or 100 µmol As/kg (0.75 mg As/kg or 7.5 mg As/kg). Control animals were given HPLC grade water alone (10 ml/kg). Mice were euthanized by exsanguinations under CO2 anesthesia between 0.25 and 24 h post dosing. Blood, liver, lung, and kidney were harvested immediately, flash frozen in liquid nitrogen and stored at –70°C until total and speciated arsenic analysis. To evaluate total flux of arsenate through metabolic pathways, separate groups of mice (n = 4 cages/dose level) received a single oral dose of arsenate (0, 10, or 100 µmol As/kg) and were placed in Nalgene® metabolism cages (Nalge Co., Rochester, NY). Mice were housed three per cage to accumulate sufficient urine for a 24-h cumulative analysis of metabolites and were acclimated to the metabolism cages for 3 days prior to dosing. The animals were maintained on the same diet with water ad libitum while in the metabolism cages.

Analytical methods.
The determination of arsenical species (iAs, MMA, and DMA) in tissue samples was performed using a Perkin Elmer 5100 atomic absorption spectrometer. Tissue samples (0.02–0.1 g) were digested using 2M phosphoric acid according Hughes et al., (2000)Go; urine samples were diluted with water and directly analyzed. The urine and digested tissue samples were assayed using a method based on the hydride generation (HG) of volatile arsines, followed by cryogenic separation and final detection of these species by atomic absorption spectrophotometry (HGAAS) (Crecelius et al., 1986Go). This method, which was done only at pH 1, generates arsines from both tri- and pentavalent arsenicals and cannot distinguish whether the arsenicals in the samples are in the trivalent or pentavalent form. Thus, the designation of iAs includes arsenite and arsenate, of MMA includes MMAIII and MMAV, and of DMA includes DMAIII and DMAV.

Total arsenic was determined by HGAAS, using a Perkin Elmer 5100 atomic absorption spectrometer equipped with a FIAS-200 flow injection system. The biological samples were completely wet digested with sequential addition of nitric, sulfuric, and perchloric acids (Cox, 1980Go). This procedure converts all arsenicals to iAs. All arsenic analysis was made using an arsenic electrodeless discharge lamp at 197.3 nm in a heated quartz cell.

Working stock solutions containing 1 mg arsenic per ml were prepared daily. Quality control for total arsenic determinations included the analysis of dried liver standard (SRM1577b) concurrently with tissues samples; we attained an accuracy of 103–105% and a 3–12% coefficient of variation. The reliability of arsenic species separation procedures was assessed by spiking control liver samples with known amounts of iAs, MMAV, and DMAV (0.25, 0.25, and 0.50 µg/g, respectively). Recoveries ranged from 89% to 111% with coefficients of variation between 3 and 11%. The estimated limits of detection were 0.012, 0.012, and 0.025 µg/g of iAs, MMA, and DMA, respectively. Trimethylated arsenicals (e.g., arsenobetaine) were not detected in experimental animal tissues. Therefore, a comparison of arsenic concentration in tissues estimated by total arsenic analysis and by summation of arsenic species was used to verify the accuracy of the analytical data.

The area under the curve (AUC) for iAs, MMA, and DMA in each tissue from 0 to 24 h was estimated using PK Solutions Version 2.06 (Summit Research Services, Montrose, CO). The AUC of the speciated arsenic from the 100 µmole As/kg as arsenate dose group was compared to data from a 100 µmole As/kg as arsenite dose group from a previous study in our laboratory (Kenyon et al., 2005Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inorganic arsenic, MMA, and DMA were detected in all tissues at various times after oral administration of arsenate. Comparison of total arsenic with the sum of species for all tissues indicated that iAs, MMA, and DMA accounted for all of the arsenic present in tissues (data not shown). The time to peak tissue levels of iAs was 1 h for blood (Fig. 1), liver (Fig. 2), lung (Fig. 3), and kidney (Fig. 4) for both dose levels. MMA behaved similarly, in that peak levels were achieved in most tissues by 1 h; the only clear exception to this general trend was the peak levels of MMA in lung in high-dose mice occurred at 2 h post dosing. The levels of iAs and MMA declined quickly after reaching their peak. This is most likely a reflection on the rapidity of methylation of arsenic to DMA as well as its rapid excretion in urine. The time to peak tissue levels of DMA was reached at 2 h in all tissues, except for lung in the 100 µmol As/kg dose group, which was reached at 4 h. The later time for peak levels of DMA is most likely a reflection of DMA being the major end product of arsenic methylation. The methylation of arsenate to DMAV requires two reduction and two methylation steps. Unlike iAs and MMA, the clearance of DMA from the tissues was more prolonged. The levels of DMA at 4 and 8 h in tissues were on the order of 4- to 100-fold greater than in blood, with the greatest difference being in the lung at 8 h.



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FIG. 1. Blood concentration time course for iAs, MMA, and DMA following a single oral dose of sodium arsenate at 10 µmol/kg (A) or 100 µmol/kg (B). Data are mean ± SD (n = 4 mice/time point).

 


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FIG. 2. Liver concentration time course for iAs, MMA, and DMA following a single oral dose of sodium arsenate at 10 µmol/kg (A) or 100 µmol/kg (B). Data are mean ± SD (n = 4 mice/time point).

 


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FIG. 3. Lung concentration time course for iAs, MMA, and DMA following a single oral dose of sodium arsenate at 10 µmol/kg (A) or 100 µmol/kg (B). Data are mean ± SD (n = 4 mice/time point).

 


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FIG. 4. Kidney concentration time course for iAs, MMA, and DMA following a single oral dose of sodium arsenate at 10 µmol/kg (A) or 100 µmol/kg (B). Data are mean ± SD (n = 4 mice/time point).

 
Data on the cumulative excretion of iAs and its methylated metabolites in urine is given in Table 1. The 24-h cumulative amount of total arsenic (inorganic + organic arsenic) excreted was 25.2 ± 3.0 µg/kg body weight (BW) in control animals, 400 ± 54.2 µg/kg BW in 10 µmole As/kg-treated animals, and 3110 ± 172 µg/kg BW in 100 µmole As/kg-treated animals. With a 10-fold increase in administered dose, there was an ~8-fold increase in the amount of arsenic eliminated in urine. As the dose was increased, the percentage of metabolites excreted as iAs increased, and the percentage of metabolites excreted as DMA decreased. This reflects either saturation or inhibition of methylation.


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TABLE 1 Urinary Excretion of Inorganic Arsenic and Its Metabolites in Mice Following Oral Administration of Arsenate

 
The AUC for iAs, MMA, and DMA in the liver, lung, and kidney were equal to or exceeded the AUC for these arsenicals in blood (Table 2). This is consistent with the observation that levels of all arsenicals in blood are low, and clearance from blood and uptake into organs is rapid. For iAs, the liver had the highest AUC at both doses of AsV, with lower AUC in lung and blood. For MMA, the kidney had the highest AUC for both doses, followed by the lung and then liver and blood. For DMA, the highest AUC was in the lung at the high dose, and the second highest AUC occurred in lung at the low dose. Kidney had the highest AUC at the low dose and the second highest AUC at the high dose for DMA. The distribution of metabolites within tissues based on AUC is noteworthy. In the blood, kidney, and liver, the distribution was roughly 40–60% iAs, 5–20% MMA, and 30–60% DMA. In contrast, the distribution in lung was 10% iAs, 10% MMA, and 80% DMA.


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TABLE 2 AUC in Tissues of Mice after Oral Administration of Arsenate or Arsenite*

 
When AUCs are compared for high equimolar (100 µmol As/kg) doses of AsV and AsIII, several striking differences are noted (Table 2). For the liver, the AUC for iAs and MMA are 2-fold and over 4-fold higher, respectively, for mice dosed orally with AsIII; whereas the AUC for DMA in liver is slightly lower for AsIII-dosed mice compared to AsV-dosed mice. Interestingly, the AUC for both iAs and MMA in kidney was 35–40% lower in AsIII- compared to AsV-dosed mice while the AUCs for DMA were more comparable. The reverse was true for lung, although the AUC for DMA was ~30% lower in AsIII-dosed mice compared to AsV-dosed mice. The relative trends for AUCs for arsenicals in blood compared to other tissues are quite similar for AsV- and AsIII-dosed mice.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study is unique in that we report speciated arsenic metabolite levels in multiple tissues across time in addition to cumulative urinary elimination data following oral administration of arsenate in the mouse. Arsenate administered orally to mice was rapidly and extensively metabolized and excreted principally in the urine. This is consistent with results in our previous studies in which the time course of urinary arsenic elimination was investigated (Hughes et al., 1994Go; Kenyon et al., 1997Go). Similar results have been reported in other studies as well (Vahter, 1981Go; Vahter and Norin, 1980Go; Vahter and Marafante, 1983Go). The study reported here clearly demonstrates that tissue levels of arsenicals are both tissue specific and dose dependent and not generally reflective of overall flux through the metabolic pathways as measured by cumulative urinary elimination.

The urine contains principally DMA (60–80% of dose) followed by 20–40% as iAs, and a small percentage as MMA (less than 4%). This is in contrast to the levels of the speciated arsenic in the tissues. Based on AUC (Table 2), there are nearly equivalent tissue doses of iAs and DMA in the blood, liver, and kidney. The main difference is that in the lung, there is a greater concentration of DMA than iAs. The concentration of MMA is lower than the other two arsenicals. Thus it appears that speciated urinary arsenic adequately reflects speciated lung arsenic levels, but not speciated arsenic levels in blood, liver or kidney. The percentage distribution of iAs, MMA, and DMA in the blood, kidney, and liver is higher for iAs and MMA, and lower for DMA compared to urine at both dose levels in this study. This trend was also observed in our repeated-dose study with arsenate administered orally to mice (Hughes et al., 2003Go). These results indicate that, although speciated urinary arsenic analysis is a useful measure of exposure and flux through the metabolic pathways (i.e., methylation capacity or efficiency), it does not adequately describe tissue distribution of arsenicals.

One limitation of the data generated in this study is that the HGAAS speciated analytical method used, done only at pH 1, does not distinguish the oxidation states of the arsenicals (i.e., iAs includes AsIII plus AsV, MMA includes MMAIII plus MMAV, and DMA includes DMAIII plus DMAV). Whenever possible, distinguishing between the pentavalent and trivalent oxidation states is highly desirable because of the differential toxicity of the various arsenical species. Other methods such as pH-selective HGAAS (Devesa et al., 2004Go), HPLC-ICP-MS (B'Hymer and Caruso, 2004Go) or HPLC-AFS (Gong et al., 2002Go) can potentially overcome this limitation, at least for measurement of urinary arsenic metabolites. However these methods are not yet sufficiently developed and validated to routinely distinguish arsenical oxidation states in tissue, as opposed to urine. Speciated urinary arsenic data where trivalent and pentavalent organoarsenic measurements are reported must also be interpreted with caution in light of a recent report of a sulfur-containing dimethylarsenic species detected in urine of sheep administered arsenosugars being mistaken for DMAIII (Hansen et al., 2004Go). Thiol-containing organoarsenicals have also been identified in the liver of rats administered methylated arsenicals intravenously (Suzuki et al., 2004Go).

In the present study, the tissue distribution of DMA is quite different from that of the other arsenic species following arsenate administration in the present study. In the kidney and liver, the maximum concentration (Cmax) of arsenic was at 1 h post dosing in the form of iAs. This was also observed in blood. In contrast, the Cmax of arsenic in the lung occurred at 2–4 h in the form of DMA. Similar results were observed following a 100 µmol As/kg oral dose of arsenite to mice in a previous study (Kenyon et al., 2005Go). This suggests that either DMA is formed in the liver from iAs and then transported to the lung and accumulates over a short time, or alternatively, that iAs is transported in the blood to the lung and then is methylated to DMA. Vahter et al. (1984)Go and Hughes et al. (2000)Go have observed preferential distribution of DMA to lung following iv administration of DMAV. This would suggest that a unique mechanism exists to sequester the dimethylated form of arsenic in lung irrespective of methylation in the lung or other organs. The mechanism for the preferential distribution of DMA to lung is not known. However, DMAV induces a lung-specific lesion (single strand breaks in DNA) in both mice and rats when administered orally as DMAV and in human lung cells treated with DMAV (Yamanaka and Okada, 1994Go); DMAV is also a promoter of tumors in mouse lung (Yamanaka et al., 1996Go; see also reviews by Kenyon and Hughes, 2001Go; Wanibuchi et al., 2004Go). Taken together, the lung-specific toxicity and lung accumulation of dimethylated arsenic suggest that evaluation of mechanistic and risk assessment implications of this phenomenon is warranted.

In our study and others (Hughes et al., 1994Go; Kenyon et al., 1999Go; Vahter 1981Go), as the oral dose of arsenate is increased, less DMA and more MMA and iAs are excreted in urine. This is also observed following oral administration of arsenite in the mouse (Kenyon et al., 2005Go; Vahter, 1981Go). This finding suggests that either saturation or inhibition of methylation is occurring. Methylation studies using rat liver cytosol support the hypothesis that arsenite is inhibiting the formation of dimethylated arsenic from monomethylated arsenic. In these studies it is observed that, with increasing initial arsenite concentration, the amount of DMA formed decreased and the time lag in DMA formation increased while MMA concentration remained constant or increased (Buchet and Lauwreys, 1985Go; Styblo et al., 1996Go). Csanaky et al. (2003)Go have investigated the mechanism of dose-dependent decrease in arsenic methylation observed in rats following arsenite administration and concluded that the most likely explanation was methyltransferase inhibition.

A comparison of 24-h cumulative urinary metabolites of mice administered equimolar dose (100 µmol As/kg) of arsenate or arsenite indicates there is a difference in the metabolic profile (Kenyon et al., 2005Go). A lower percentage of the dose was excreted in urine as DMA (60%) in arsenate-treated mice compared to arsenite-treated mice (80%). MMA excretion was low (6% or less) for both AsIII and AsV. A greater percentage of the dose was excreted as iAs in arsenate-treated mice (40%) compared to arsenite-treated mice (15%) (Kenyon et al., 2005Go). This indicates that arsenite is more easily methylated than arsenate, perhaps because of greater uptake into tissues that methylate arsenic and more rapid excretion of pentavalent methylated arsenic. Styblo et al. (1995)Go have shown in rat hepatic cytosol that 90% of arsenite is methylated to dimethyl arsenic, whereas only 40% of arsenate is methylated during a 90-min incubation. This suggests that either reduction of arsenate to arsenite or MMAV to MMAIII may also be a rate-limiting step, although Zakharyan and Aposhian (1999)Go have reported that the rate-limiting step is MMAV to MMAIII reduction.

A comparison of the distribution of arsenicals in tissues of arsenate- and arsenite-treated animals again demonstrates that urinary arsenic speciation does not adequately reflect tissue distribution of arsenic species. There is a trend of slightly less DMA and more iAs and MMA in arsenite-dosed mice compared to arsenate-dosed mice. The largest difference is in the liver. In arsenate-treated mice, approximately 60% of the arsenic was in the form of iAs, whereas it was 70% in the arsenite-treated mice. This may be a reflection of the binding of arsenic (arsenite directly and arsenate after reduction to arsenite) to hepatic macromolecules.

The relationship of exposure dose and tissue concentration of parent chemical and metabolites is a critical issue in cases where toxicity may be mediated by a metabolite or parent chemical and metabolite acting together. This has emerged as an issue for iAs, because both its trivalent and pentavalent methylated metabolites have unique toxicities and the trivalent methylated metabolites of arsenic exhibit greater potency than AsIII for some endpoints. In this study, the time-course tissue distribution of iAs and its methylated metabolites was determined in blood, liver, lung, and kidney, and cumulative urinary excretion measured in female B6C3F1 mice given a single oral dose of 0, 10, or 100 µmol/kg sodium arsenate. This study clearly demonstrates that tissue levels of arsenicals are both tissue specific and dose dependent and not generally reflective of overall flux through the metabolic pathways as measured by cumulative urinary elimination, except for possibly the lung. Thus, failure to adequately characterize their target tissue dosimetry can result in erroneous risk estimates. These studies provide useful data for physiologically based pharmacokinetic model development to improve risk assessment.


    NOTES
 
Portions of this work were presented at the Society of Toxicology 2001and 2003 meetings. This manuscript has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.


    ACKNOWLEDGMENTS
 
Dr. Luz Maria Del Razo was supported in part by a CONACYT-Mexico fellowship. We thank Brenda Edwards, Carol Mitchell, and Karen Herbin-Davis of the U.S. EPA for their assistance with animal handling and analytical work.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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