Disposition of Inorganic Arsenic
This chapter reviews the data regarding the absorption, biotransformation, distribution, and elimination of arsenic in animals and humans. Physiologically based pharmacokinetic (PB-PK) models that incorporate that information are described in the section Kinetic Model. The implications of the subcommittee's conclusions for a risk assessment for arsenic in drinking water are presented in the section Summary and Conclusions.
When ingested in dissolved form, inorganic arsenic is readily absorbed. About 80-90% of a single dose of arsenite As(III) or arsenate As(V) was absorbed from the gastrointestinal tract of humans and experimental animals (Pomroy et al. 1980; Vahter and Norin 1980; Freeman et al. 1995). A much lower degree of gastrointestinal absorption was reported for arsenic-contaminated soil (Freeman et al. 1995), although the form of arsenic in the soil, as well as the type of soil, can be assumed to influence the degree of arsenic absorption. Also, arsenic compounds of low solubility (e.g., arsenic selenide) (Mappes 1977), arsenic trisulfide and lead arsenate (Marafante et al. 1987), and gallium arsenide (Webb et al. 1984; Yamauchi et al. 1986) are absorbed much less efficiently than is dissolved arsenic. There is a lack of data on the bioavailability of inorganic arsenic in various types of foods.
No controlled studies have been conducted on the rate of absorption of inorganic arsenic through intact human skin. However, reported systemic toxicity in persons having extensive acute dermal contact with solutions of inorganic arsenic indicates that skin can be a route of exposure (Hostynek et al. 1993). In vitro studies in which water solutions of radiolabeled arsenate
were topically applied to human skin or the skin of rhesus monkeys showed that about 2-6% of the applied arsenic was absorbed in 24 hr (Wester et al. 1993). Similar in vitro studies using dorsal skin of mice showed a much higher absorption; 30% of the applied dose of radiolabeled arsenate in aqueous solution (100-200 ng/L) was absorbed in 24 hr (Rahman et al. 1994). A large percentage, on average 60-90 %, of the absorbed arsenic was retained in the skin. That result indicates that inorganic arsenic can bind externally to skin and hair. Rapid binding of 74 As to the skin and epithelium of the upper gastrointestinal tract in the marmoset fetus has also been observed 8 hr after maternal exposure to 74As-arsenite (Lindgren et al. 1984). Taken together, those results indicate a low degree of systemic absorption of arsenic via the skin. Some further information on the skin absorption of arsenic in humans may be obtained from a study in Fairbanks, Alaska, where arsenic was found in home water at 345 µg/L (Harrington et al. 1978). One group of people who drank bottled water only but used the arsenic-rich water for other purposes had about the same low concentrations of the sum of arsenic metabolites in urine (average 43 µg/L) as people with less than 50 µg/L in their home water (average 38 µg/L in urine), indicating a low degree of skin absorption. However, the hair arsenic concentrations were clearly elevated in the group drinking bottled water (5.7 µg/g compared with 0.43 µg/g in the low-arsenic-water group), suggesting that arsenic is bound externally to hair and probably also to skin during washing with arsenic-rich water.
In this section, arsenate reduction and arsenite methylation are described. Species differences are reviewed, as are other factors influencing the metabolism of arsenic. Variations in arsenic methylation in humans are reviewed in more detail in Chapter 7.
Arsenate Reduction and Arsenite Methylation
In humans and in most experimental animals, inorganic arsenic is methylated to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). Compared with inorganic arsenic, the methylated metabolites are less reactive with tissue constituents, less acutely toxic, less cytotoxic, and more readily excreted in the urine (Buchet et al. 198 1a; Vahter and Marafante 1983; Vahter et al. 1984; Yamauchi and Yamamura 1984; Marafante et al. 1987; Moore et al. 1997; Rasmussen and Menzel, 1997; Concha et al. 1998a; Hughes and
Kenyon 1998; Sakurai et al. 1998). In addition, experimental studies showed that inhibition of the methylation reactions results in increased tissue concentrations of arsenic (Marafante and Vahter 1984; Marafante et al. 1985).
In the 1930s, Challenger found that microorganisms grown in the presence of arsenite, MMA, or DMA released trimethylarsine (Challenger 1945). Essentially the same sequence of alternating reduction and methylation reactions was postulated for various mammals exposed to inorganic arsenic (Challenger 1945). However, considerable variation in methylation products is found among species. For example, DMA is the major end point of arsenic biomethylation in most mammals, while in many microorganisms, trimethylarsine is the end product. In mice, hamsters, and humans exposed to DMA, a small fraction was further methylated and excreted in the urine as trimethylarsine oxide (Marafante et al. 1987). However, the formation of trimethylarsine or its oxide has not been demonstrated following exposure to inorganic arsenic.
As noted in Chapter 3, methylation of inorganic arsenate to DMA involves alternating reduction of pentavalent arsenic to trivalent arsenic and addition of methyl groups (Vahter and Envall 1983; Cullen et al. 1984; Marafante et al. 1985; Vahter and Marafante 1988; Buchet and Lauwerys 1988; Hirata et al. 1990; Thompson 1993). Although all the steps and mechanisms in the arsenic biotransformation have not been elucidated, experimental animal studies performed in vivo or with animal tissue preparations in vitro indicate that the methylation takes place by transfer of methyl groups from S-adenosylmethionine (SAM) to arsenic in its trivalent oxidation state (Marafante and Vahter 1984; Buchet and Lauwerys 1985; Marafante et al. 1985; Styblo et al. 1995, 1996; Zakharyan et al. 1995). Probably, glutathione (GSH) plays an important role in the reduction of As(V) to As(III). The produced As(III) can form a complex with GSH (Delnomdedieu et al. 1994a,b). Also, cysteine or dithiothreitol (DTT) can reduce pentavalent arsenic, as shown in studies with purified rabbit-liver enzyme preparations (Zakharyan et al. 1995) or rat-liver cytosols (Buchet and Lauwerys 1985; Styblo et al. 1996).
Studies with mice, rabbits, and marmoset monkeys showed that a substantial fraction of absorbed As(V) is rapidly reduced, probably mainly in the blood, to As(III) (Vahter and Envall 1983; Vahter and Marafante 1985; Marafante et al. 1985), most of which is then methylated to MMA and DMA (Marafante et al. 1985). As(V) might also be reduced in the stomach or intestine, but quantitative experimental data are not available. Because of the rapid reduction, chronic exposure to arsenite and arsenate will result in fairly similar metabolite distribution in the body. However, the distribution pattern will differ for the two forms if the reducing capacity is exceeded by acute high-dose exposure (Vahter 1981; Lindgren et al. 1982). If the newly formed
trivalent arsenic is not completely methylated, the reduction of As(V) to As(III) results in enhanced retention in most tissues, because trivalent arsenic is more reactive with tissue constituents than is pentavalent arsenic (Vahter and Marafante 1983; Bogdan et al. 1994; Styblo et al. 1995).
Following ingestion of MMA(V) by humans, only about 10% is further methylated and excreted as DMA in the urine (Buchet et al. 1981a). Similar results were obtained in vitro with rat-liver cytosols, while more than 90 % of added MMA in reduced form, MMA(III), was further methylated to DMA (Styblo et al. 1995). That result indicates that MMA is not reduced as easily as arsenate or that there is a slow cellular uptake of MMA in vivo (Delnomdedieu et al. 1995; Mann et al. 1996a).
The kinetics of arsenic methylation in vivo has not been completely elucidated. In rabbits exposed to inorganic arsenic, DMA appeared in the liver before appearing in other tissues (Marafante et al. 1985). Further support for the finding that the liver is an important initial site of arsenic methylation is obtained in studies conducted in experimental animals and humans. Orally administered inorganic arsenic, most of which initially passes through the liver following absorption, was found to be methylated more efficiently than inorganic arsenic administered subcutaneously or intravenously (Charbonneau et al. 1979; Vahter 1981; Buchet et al. 1984). Also the fact that the methylation of arsenic injected in patients with end-stage liver disease improved markedly with liver transplantation suggests that the liver plays an important role in arsenic metabolism (Geubel et al. 1988). The site of methylation might also depend on the rate of reduction of As(V) to As(III). Studies on isolated rat hepatocytes showed that arsenite, but not arsenate, is readily taken up and methylated by the liver cells (Lerman et al. 1983). That might occur because the trivalent inorganic arsenic is in non-ionized form (arsenous acid) at physiological pH, and the pentavalent form is ionized (see Chapter 3). On the other hand, studies with kidney slices showed that about five times more DMA is produced from arsenate than from arsenite (Lerman and Clarkson 1983), indicating that the arsenate that is not initially reduced to arsenite can be taken up by the kidney cells, reduced and methylated to DMA intracellularly, and then excreted in the urine. Reabsorption and reduction of arsenate in the kidney tubule were demonstrated in dogs (Ginsburg 1965).
In vitro studies investigating the methylation capacity of different tissues had varying results. Buchet and Lauwerys (1985) reported that the methylating capacity of red blood cells, brain, lung, intestine, and kidneys of rats was insignificant compared with that of the liver. In vitro studies using arsenite methyltransferases from mouse tissues showed that the highest amount of methylating activity is in the testes, followed by kidney, liver, and lung (Healy et al. 1998). The amounts of methyltransferases vary in different
tissues and animal species (Aposhian 1997). Although the methylating capacity of tissues in vitro does not reflect in vivo methylation, when the kinetics of the arsenic species plays an important role, the results indicate that arsenite initially bound to tissue constituents can be methylated and released. That could explain the observed slow elimination phase that follows the initial rapid phase; the slow elimination phase might involve the continuous release of arsenic from most binding sites (Marafante et al. 1981; Vahter and Marafante 1983).
In vitro studies using rat-liver preparations showed that the methylating activity is localized in the cytosol and that SAM is the main methyl donor (Buchet and Lauwerys, 1985; Zakharyan et al. 1995; Styblo and Thomas 1997). Added vitamin B12, coenzyme B12, and methylcobolamin could also act as methyl donors, and the latter could produce MMA even in the absence of enzymatic activity. Research on the purification and characterization of arsenic methyltransferases (Zakharyan et al. 1995, 1996) indicated that the rabbit arsenite and MMA methyltransferase activities are in the same protein. Using the 2,000-fold purified protein, the investigators found no evidence that the activities were on different proteins (Zakharyan et al. 1995). SAM was used as the methyl source, as previously shown by in vivo studies and in vitro studies using tissue cytosol. The arsenite and the MMA methyltransferase had a molecular mass of 60 kDa, as determined by gel-size-exclusion chromatography but had different pH optima and different saturation concentrations for their substrates. Neither arsenate nor selenate, selenite, or selenide was methylated by the purified enzyme preparations.
There are major species differences in the biotransformation of inorganic arsenic (Vahter 1994). A number of studies, in which the metabolites of inorganic arsenic in human urine have been speciated, consistently show average values of 10-30% inorganic arsenic, 10-20% MMA, and 55-75% DMA (for a review, see Hopenhayn-Rich et al. 1993). Those results were found in human subjects exposed to inorganic arsenic in the general environment and in those exposed at work. However, in recent studies of people exposed to arsenic via drinking water in northern Argentina, urinary arsenic consisted of only 2% MMA on average (Vahter et al. 1995a; Concha et al. 1998a). Variations in arsenic methylation in humans is reviewed in more detail in Chapter 7.
Many experimental animals excrete less MMA and more DMA in the urine than do humans. Mice and dogs methylate inorganic arsenic efficiently,
and in general, more than 80% of the administered dose is excreted, mainly as DMA, in the urine within a few days (Charbonneau et al. 1979; Vahter 1981). The rat also methylates inorganic arsenic efficiently, but a major portion of the DMA produced is retained in the erythrocytes (Odanaka et al. 1980; Lerman et al. 1983), giving rise to a slow urinary excretion of DMA and a tissue-distribution pattern that is different from that in most other species (Vahter et al. 1984). In addition, the rat shows an extensive biliary excretion of arsenic, about 800 and 37 times more than the dog and rabbit, respectively (Klaassen 1974).
With respect to arsenic methylation, the rabbit (Marafante et al. 1981; Vahter and Marafante 1983; Maiorino and Aposhian 1985) and the hamster (Charbonneau et al. 1980; Yamauchi and Yamamura 1984, 1985; Marafante and Vahter 1987) are more similar than other investigated animal species to humans, although more DMA and less MMA is excreted by rabbits and hamsters than by humans. The Flemish giant rabbit (De Kimpe et al. 1996) and a New Zealand rabbit (Bogdan et al. 1994) were found to excrete MMA in amounts similar to those in humans.
The two animal species that were first shown not to methylate inorganic arsenic, the marmoset monkey (Vahter et al. 1982; Vahter and Marafante 1985) and the chimpanzee (Vahter et al. 1995b), in general have a metabolism most similar to that of humans. The cynomolgus monkey, on the other hand, seems to methylate inorganic arsenic well (S.M. Charbonneau, Health and Welfare Canada, Ottawa, Ont., personal commun., 1983; cf. Vahter 1983). Another unique feature of the marmoset monkey is that it accumulates arsenic in the liver, apparently firmly bound to the rough microsomal fraction (Vahter et al. 1982). The first phase of elimination has a fairly short half-time. In the second phase, as much as 70% of the administered dose is eliminated at a very slow rate.
In the chimpanzee, excretion also seems to be biphasic, and the half-time of the second phase is similar to that observed in the marmoset; however, much less of the total administered dose is eliminated. In spite of the lack of methylation of arsenic in the chimpanzee, about 50% of an intravenous dose was excreted within 2 to 4 days in the urine (Vahter et al. 1995b). That is similar to that in humans, who excrete about half of a low dose of arsenite or arsenate in the urine within about 4 days (Tam et al. 1979; Pomroy et al. 1980; Buchet et al. 1981b), indicating that other factors influence the tissue retention and excretion of arsenic.
Arsenite methyltransferase activity, tested by incubation of liver preparations with arsenite in vitro, has been detected in the liver of the rabbit, rat, mouse, hamster, pigeon, and rhesus monkey but not in the liver of the marmoset monkey, tamarin monkey, squirrel monkey, chimpanzee, and guinea pig
(Zakharyan et al. 1995, 1996; Healy et al. 1997). With human liver preparations, no methylated arsenic metabolites were detected. As humans do methylate inorganic arsenic in vivo, the reason for the negative in vitro test is not known. In the case with marmoset monkeys and chimpanzees the negative in vitro test is in concordance with in vivo studies showing no methylation of inorganic arsenic in those animals (Vahter et al. 1982; Vahter et al. 1995b).
The rhesus monkey, hamster, rat, mouse, and pigeon, have ample amounts of such methyltransferase activity. In decreasing order, the species with liver arsenite methyltransferase activity are the pigeon, rhesus monkey, mouse, hamster, rabbit, marmoset monkey, squirrel monkey, and tamarin monkey. In addition, when guinea pigs were injected intraperitoneally with radioactive arsenate, five of six guinea pigs did not have methylated arsenic species in their urine (Healy et al. 1997). The sixth animal had minimal but measurable amounts of DMA in its urine. Analysis of liver cytosol showed that the guinea pigs were deficient in liver arsenic methyltransferase activities (Healy et al. 1997). All the species had ample arsenate reductase activity, however.
Factors Influencing the Metabolism of Arsenic
It is likely that factors influencing the methylation of arsenic can modify the tissue retention and toxicity of arsenic, because the biomethylation of inorganic As(III) produces metabolites that have low reactivity toward most tissues and that are readily excreted in the urine. This section describes the experimental evidence for effects on the methylation of arsenic by such factors as the chemical form and dose of arsenic absorbed protein binding, nutrition, and genetic polymorphism. The observed variation in human methylation of arsenic in relationship to dose, sex, ethnicity, and recreational habits is discussed in Chapter 7.
In some experimental animal studies, exposure to arsenite resulted in a higher degree of methylation and more DMA in the urine than did exposure to arsenate (Vahter 1981; Vahter and Marafante 1983). However, in spite of the fact that the reduction of arsenate is not complete, the total amount of arsenic excreted in the urine is slightly higher following arsenate exposure than following arsenite exposure, as shown by experimental studies on human volunteers (Pomroy et al. 1980; Buchet et al. 1981a). Most likely, the reason for this is that As(V) is less reactive with tissue constituents and more readily excreted in the urine than is As(III) (Vahter and Marafante 1983). The differences in retention of arsenic following exposure to arsenate and arsenite decrease with decreasing dose (Vahter 1981), because a large part of the arsenate is reduced rapidly following absorption.
Studies on mice showed that as the dose of inorganic arsenic increases substantially, the methylation rate decreases and the tissue concentrations of arsenic increase (Vahter 1981; Hughes et al. 1994). Also, in humans acutely intoxicated by very high doses of inorganic arsenic, there is a marked delay in the urinary excretion of DMA (Mahieu et al. 1981; Foà et al. 1984). In one case of attempted suicide, a person ingested about 3 g of As2O3 in water and was admitted to the hospital 2 hr later (Foà et al. 1984). The relative percentage of urinary DMA increased from about 30% on the 8th day to 78 % on the 13th day after ingestion. In three persons who ingested 100-500 mg of arsenic in the form of As2O3, which was mistakenly used instead of sugar, urinary DMA increased from about 10% the first days after ingestion (blood arsenic concentrations of 190-800 µg/L) to about 75% after a week (Mahieu et al. 1981). However, in the case of exposure to arsenic via drinking water, even at very high arsenic concentrations, the methylation of arsenic seems to be relatively unaffected by the concentration of the dose. According to an abstract of a study by Kosnett and Becker (1988), following subacute exposure to drinking water containing arsenic at a concentration of 25,000 µg/L, a 36-year-old man yielded a urinary arsenic collection containing 6,025 µg per 24 hr, 26% as inorganic arsenic and 74% as methylated metabolites. A further discussion of the dose-dependence of arsenic methylation in humans exposed via drinking water is presented in Chapter 8.
Results from in vitro studies suggested that the delay in urinary excretion of DMA might occur because of the high tissue concentrations of arsenite inhibit the methyltransferase catalyzing the second methylation step (Buchet and Lauwerys 1985). Methylation capacity might also have been overloaded by the high tissue concentrations of As(III). However, in both human studies, BAL was administered for several days, which might have influenced the methylation of arsenic (see below).
Protein binding is another factor in arsenic mechanism. Bogdan et al. (1994) found three arsenite-binding proteins in rabbit liver. They were 450 kilodaltons (kDa), 100 kDa, and less than 2,000 kDa in size. Inorganic arsenite was firmly bound to them. The affinity of arsenite for those proteins was 20 times greater than that for arsenate.
Experimental studies on mice and rabbits showed that inhibition of SAMdependent methylation reactions by treatment with periodate-oxidized adenosine, which inhibits the metabolism of S-adenosylhomocysteine, results in decreased methylation and increased tissue concentrations of arsenic (Marafante and Vahter 1984; Marafante et al. 1985). That finding indicates that a low tissue concentration of SAM (e.g., as a result of low intake of precursors to SAM) might give rise to a low rate of methylation of arsenic. In fact, studies on rabbits fed diets with low amounts of methionine, choline,
or proteins showed a marked decrease in urinary excretion of DMA, accompanied by a 2-3 times increase in tissue concentrations of arsenic, especially in the liver (Vahter and Marafante 1987). A decrease in urinary excretion of arsenic was also observed in mice fed a choline-deficient diet (Tice et al. 1997). In particular, arsenic concentrations were increased in liver microsomes of the rabbits on the methyl-deficient diet (Vahter and Marafante 1987), a result similar to that seen in the marmoset monkeys (Vahter et al. 1982). Thus, nutritional factors might influence the subcellular distribution of arsenic.
The methylation of arsenic appears to be affected by hepatic disease. Various liver diseases (i.e., alcoholic cirrhosis, chronic hepatitis, homochromatosis, postnecrotic cirrhosis, steatosis, and biliary cirrhosis) decreased the proportion of MMA (in relationship to the total urinary excretion of metabolites of inorganic arsenic) and increased the proportion of DMA in urine following injection of a single dose of sodium arsenite (Buchet et al. 1984; Geubel et al. 1988). The overall effect was a more efficient methylation of the injected arsenic in individuals with liver disease than in healthy controls or controls with other types of diseases.
The effect of the chelating agent 2,3-dimercapto-1-propanesulfonic acid (DMPS; 300 mg of Dimaval given by mouth after an overnight fast) on the urinary arsenic metabolite pattern was studied in people living in the Atacama desert in northeastern Chile, where the concentration of arsenic in drinking water is 600 µg/L (Aposhian et al. 1997). During the 2-hr period following administration, the content of metabolites of inorganic arsenic in urine consisted of 20-22% inorganic arsenic, 42% MMA, and 37-38% DMA. The usual range of MMA in human urine is 10-20% and that of DMA 60-80%. A similar increase in the percentage of MMA was found in the control subjects exposed to arsenic at 20 µg/L of drinking water. DMPS increased the total urinary arsenic excretion by about 5-6 times during the 6-hr period after administration. The mechanism for the increase in MMA excretion is not known.
In vitro studies on arsenic methylation in rat-liver cytosol showed that chelating agents, such as DMSA and DMPS (0.05-0.5 mM), almost completely inhibit the methylation of inorganic arsenic to DMA (Buchet and Lauwerys 1985, 1988). Addition of DTT (dithiothreitol) and 2-mercaptoethanol of up to 0.5 mM stimulated in vitro methylation, but resulted in inhibition at higher concentrations. Also, EDTA (1 mM) was shown to inhibit preferentially DMA formation from 74As-As(III) (carrier-free) in vitro (Styblo and Thomas 1997). Possibly, the administration of chelating agents results in an inhibition of the second step of the methylation of the inorganic arsenic released from tissues, giving rise to more MMA being excreted in the urine.
Transportation, Distribution, And Elimination
In this section, the transportation of arsenic in blood, its distribution in tissues, and its elimination from the body are described.
Transportation in Blood
Absorbed arsenic is transported in the blood, mainly bound to sulfhydryl (SH) groups in proteins and low-molecular-weight compounds such as glutathione (GSH) and cysteine, to the organs in the body. Formation of complexes between trivalent arsenicals and GSH, probably mainly in the form of As(GS)3, has been demonstrated in water solutions (Scott et al. 1993; Delnomdedieu et al. 1994a), rabbit erythrocytes (Delnomdedieu et al. 1994b), and rat bile (Anundi et al. 1982). However, As(III) can be transferred easily from the As(GS)3 complex to binding sites of higher affinity. Recently, inorganic arsenic was reported to be the main form of arsenic bound to serum proteins in patients on continuous ambulatory peritoneal dialysis, and transferrin was the main carrier (Zhang et al. 1997, 1998a,b).
Most of the arsenic in blood is rapidly cleared, following a three-exponential clearance curve (Mealey et al. 1959; Pomroy et al. 1980). The majority of arsenic in blood is cleared with a half-time of about 1 hr. The half-times of the second and third phases are about 30 and 200 hr, respectively. Experimental data on animals and data on patients with uremia indicate that the concentration of arsenic in red blood cells is severalfold that in plasma at low or background exposure concentrations but is close to onefold at increased blood concentrations (Lindgren et al. 1982; Versieck 1985; De Kimpe et al. 1993). The ratio between plasma and the red blood cells might also depend on the exposure form of arsenic; studies on rabbits found that As(III) is more easily taken up by erythrocytes than is As(V), MMA, or DMA (Delnomdedieu et al. 1995). Early studies on healthy individuals with no known exposure to arsenic indicate similar concentrations (about 2.5 µg/L) in plasma and whole blood (Heydorn 1970). People from the area in Taiwan with arsenic-rich water had about 15 µg/L in plasma and 22 µg/L in whole blood. Patients with blackfoot disease and their families had about 30 µg/L in plasma and 60 µg/L in whole blood (Heydorn 1970).
Arsenic concentrations were found to be significantly higher in the serum and erythrocytes of chronic hemodialysis patients compared with controls (De Kimpe et al. 1993). Serum had a median arsenic concentration of 12 µg/L versus 0.38 µg/L in controls, and erythrocytes had a median of 9.5 µg/L
versus 3.2 µg/L in controls. A single hemodialysis treatment did not change the arsenic concentrations. In a similar study, Zhang et al. (1996) reported a mean total arsenic concentration of 5.1 µg/L in nonhemodialysis patients and 6.5 µg/L in hemodialysis patients, compared with 0.96 µg/L in a control group of healthy subjects. DMA and arsenobetaine (AsB) were the major arsenic species in serum, the mean values being about 1 µg/L for DMA and 3.5 µg/L for AsB. Serum concentrations of inorganic arsenic and MMA were below the detection limits. In the control group, serum arsenic concentrations were too low for speciation. Hemodialysis treatment removed 68% of total arsenic in serum and 16% in erythrocytes. The efficiency was similar for DMA and AsB.
There are major species differences in the half-time of arsenic in blood. In the rat, arsenic is retained in the blood considerably longer than in other species because of the accumulation of DMA in the red blood cells, apparently bound to hemoglobin (Odanaka et al. 1980; Lerman and Clarkson 1983; Vahter 1983; Vahter et al. 1984). The accumulation of arsenic in the rat erythrocytes was first reported more than 50 years ago (Hunter et al. 1942), although at that time DMA was not known to be the main form of arsenic retained. Lanz and co-workers (1950) reported that the cat also had higher concentrations of arsenic in the blood than most other species, although not as high as the rat. Whether it is DMA that accumulates in the red blood cells of the cat is not known.
Even though inorganic arsenic is not methylated in the chimpanzee, the clearance of arsenic from the plasma, following a single intravenous dose of 73 As-arsenate, was shown to be fast, with a half-time of about 1 hr (Vahter et al. 1995b). The elimination from red blood cells was slower, with a half-time of about 5 hr. Essentially, all 73 As in the plasma was ultrafiltrable, indicating a low degree of binding to high-molecular-weight proteins (above 25,000 daltons). Those findings indicate that the rate of clearance of arsenic from blood might be affected by factors other than methylation.
In the body, As(III) is mainly bound to SH groups. In particular, As(III) forms high-affinity bonds with vicinal thiols, as demonstrated with lipoic acid and DMSA (Cullen and Reimer 1989; Delnomdedieu et al. 1993). Experimental animal studies found that the binding of arsenic is mainly to highmolecular-weight proteins in various tissues; however, arsenic is continuously released from most intracellular binding sites over time following exposure (Marafante et al. 1981; Vahter et al. 1982). Probably, As(III) is bound to
proteins before undergoing subsequent methylation-reduction reactions. Whether the protein is methyltransferase is not known. Compounds of the type Me2AsSR, formed following addition of the second methyl group, are easily oxidized to DMA (Cullen and Reimer 1989), which is then excreted in the urine. However, the stability might vary, and DMA complexes have been detected in urine (Marafante et al. 1987). A very stable complex appears to be formed between DMA and hemoglobin in the rat (Lerman et al. 1983). In vitro studies indicate the formation of mixed protein hemoglobin-GSH complex with As(III) (Winski and Carter 1995).
In experimental studies on mammals exposed to inorganic arsenic, the tissues with the longest retention of arsenic were skin, hair, squamous epithelium of the upper gastrointestinal tract (oral cavity, tongue, esophagus, and stomach wall), epididymis, thyroid, skeleton, and the lens of the eye (Lindgren et al. 1982, 1984; Vahter et al. 1982). Except for the skeleton, all the tissues mentioned contained higher concentrations of arsenite than arsenate shortly after administration of either. Following administration of arsenate to mice, immediate accumulation and long-term retention of arsenic were observed in the calcified areas of the skeleton, probably reflecting the substitution of phosphate by arsenate in the apatite crystals in bone because of the chemical similarities between arsenate and phosphate (see Chapter 3). However, the differences in distribution in relationship to the exposure form of arsenic decreased over time following exposure. A few days after the administration of arsenic, the distribution pattern was essentially the same irrespective of the form of arsenic administered. The similar pattern of distribution following administration of fairly low doses of arsenate and arsenite might be explained by the rapid in vivo reduction of As(V) to As(III), as demonstrated in various mammalian species (Ginsburg 1965; Vahter and Envall 1983; Marafante et al. 1985).
Most likely, inorganic arsenic, mainly As(III), is the form bound to SH groups of keratin in the skin, hair, oral mucosa, and esophagus. That was evident from the distribution pattern of arsenite in the marmoset monkey, which does not methylate inorganic arsenic (Vahter et al. 1982). Furthermore, chemical inhibition of the transfer of methyl groups from S-adenosylmethionine in mice and rabbits resulted in increased arsenic concentrations in most tissues, especially the skin (Marafante and Vahter 1984). The half-time in skin seems to be more than 1 month (Du Pont et al. 1941). Scott (1958) used neutron-activation analysis to assess the arsenic content of benign and malignant skin lesions of 14 patients exposed at least 4 years earlier to several years of inorganic arsenical medication. Arsenic content of the biopsied skin ranged from 0.8 to 8.9 ppm, and on average exceeded the arsenic content of normal skin and malignant skin lesions from six subjects with no history of
arsenic intake (range 0.4 to 1.0 ppm). In contrast to the rodents, the marmoset monkey showed an accumulation of arsenic in the testes, mainly localized to the spermatogenetic epithelium (Vahter et al. 1982). Accumulation of arsenic in the liver of marmosets was also pronounced.
The tissue retention of MMA and DMA is much lower than that of inorganic arsenic. Following administration of 74As-DMA to mice, its concentrations decreased rapidly in most tissues (Vahter et al. 1984). The tissues with the longest retention of DMA were the lungs, thyroid, intestinal mucosa, and the lens of the eyes. Accordingly, there was no localization of arsenic in the thyroid and ocular lens of the marmoset monkey, which did not methylate the administered inorganic arsenic (Vahter et al. 1982).
In human subjects exposed to normal environmental concentrations of arsenic, the hair and nails had the highest concentrations of arsenic (0.02-1 mg/kg of dry weight), and fairly high concentrations were found in the skin and lungs (0.01-1 mg/kg of dry weight) (Liebscher and Smith 1968; Cross et al. 1979; Das et al. 1995). Thus, arsenic appears to concentrate in tissues with a high content of cysteine-containing proteins. The arsenic concentrations in skin and kidneys of adults in Japan were reported to be 2-3 times higher than those in 1-year-old children (Kadowaki 1960). In West Bengal, India, where people are exposed to arsenic in drinking water (district average arsenic values of 200-700 µg/L), arsenic concentrations in skin scale, hair, and nails were 1.9-5.5, 3.6-9.6, and 6.1-23 mg/kg of dry weight, respectively (Das et al. 1996). However, the concentration of arsenic in skin, hair, and nails that is due to external contact with arsenic in water is not known.
Experimental animal studies showed that inorganic arsenic, trivalent as well as pentavalent, and the methylated metabolites cross the placenta during the entire gestational period (Lindgren et al. 1984; Hood et al. 1982, 1987). Administration of arsenite showed somewhat less placental transfer of arsenic in a marmoset monkey (known not to methylate arsenic) than in mice (Lindgren et al. 1984). The tissue distribution of arsenic was similar in fetus and mother. In a study of pregnant women living in a village in northwestern Argentina where drinking water contains arsenic at about 200 µg/L, arsenic concentrations were about as high in cord blood (on average 9 µg/L) as in maternal blood, indicating that arsenic readily reaches the human fetus (Concha et al. 1998b). The placentas also had clearly increased arsenic concentrations. However, more than 90% of the arsenic in urine and plasma of the newborns and their mothers (at the time of delivery) was in the form of DMA, indicating that arsenic methylation is induced during pregnancy and that the fetus is exposed mainly to DMA, at least in late gestation. The fetal toxicity of arsenic, however, remains to be elucidated. In pregnant women with no known arsenic exposure, the concentration of arsenic in cord blood
was found to be about the same (average 2-3 µg/L) as that in maternal blood (Kagey et al. 1977). Increased concentrations of arsenic have been detected in the placentas of women living near smelters (Tabacova et al. 1994).
The studies of women living in northwestern Argentina indicated a low degree of arsenic excretion in human breast milk (Concha et al. 1998c). The average concentration of arsenic in milk was 2 µg/kg, compared with 10 µg/L in maternal blood and 320 µg/L in maternal urine. Breast feeding was shown to decrease the concentrations of arsenic in the urine in the newborn child (Concha et al. 1998c). The arsenic concentrations found in human breast milk were slightly higher than the lowest concentrations reported in previous studies (Byrne et al. 1983; Dang et al. 1983; Parr et al. 1991). The arsenic exposure of the women in those studies was not reported, and the form of arsenic in breast milk is not known. An average breast-milk arsenic concentration of about 19 µg/kg was reported from the Philippines (Parr et al. 1991), indicating that organic arsenic compounds originating from seafood might be excreted in the milk.
In rabbits and mice exposed to radiolabeled arsenic, a major part of the arsenic in liver, kidneys, and lungs was present in the nuclear and soluble fractions (Marafante et al. 1981; Marafante and Vahter 1984). A different intracellular distribution was observed in the liver of the marmoset monkey, which is unable to methylate inorganic arsenic (Vahter et al. 1982; Vahter and Marafante 1985). Almost 50% of the arsenic was present in the microsomal fraction, apparently in a very strong association, leading to a long half-time of arsenic in the liver. Chemically induced inhibition of arsenic methylation did not change the intracellular binding of arsenic in rabbit tissues (Marafante and Vahter 1984; Marafante et al. 1985). However, in rabbits fed diets with low contents of methionine, choline, or proteins, leading to a decrease in arsenic methylation and an increase in tissue concentrations of arsenic, especially in the liver, there was an increase in arsenic in the microsomal fraction of the liver (Vahter and Marafante 1987) similar to that in the marmoset monkey. Mice fed a choline-deficient diet showed a similar decrease in the urinary excretion of DMA, but the arsenic-induced DNA damage shifted from the liver to the skin (Tice et al. 1997). These studies indicate that nutritional status might influence the intracellular distribution of arsenic and possibly its toxic effects.
The major route of excretion of most arsenic compounds is via the urine. Following exposure to inorganic arsenic, the biological half-time is about 4 days. It is slightly shorter following exposure to As(V) than to As(III) (Yamauchi and Yamamura 1979; Tam et al. 1979; Pomroy et al. 1980; Buchet et al. 1981a). In six human subjects who ingested radiolabeled 74 As-arsenate, 38% of the dose was excreted in the urine within 48 hr and 58% within 5 days (Tam et al. 1979). The results indicate that the data were best fit to a three-compartment exponential function, with 66 % excreted with a half-time of 2.1 days, 30% with a half-time of 9.5 days, and 3.7% with a half-time of 38 days (Pomroy et al. 1980). In three subjects, each of whom ingested 500 µg of arsenic in the form of arsenite in water, about 33 % of the dose was excreted in the urine within 48 hr, and 45% within 4 days (Buchet et al. 1981a). The methylated metabolites MMA and DMA are excreted in the urine faster than the inorganic arsenic. In humans, about 78% of MMA and 75% of DMA were excreted in the urine within 4 days of ingestion of the dose (Buchet et al. 1981a). Similar results were reported for mice in which the half-time of MMA and DMA was about 1 hr (Hughes and Kenyon 1998). The 24-hr whole-body retention was about 2% of the dose.
Although absorbed arsenic is removed from the body mainly via the urine, small amounts of arsenic are removed via other routes (e.g., skin, sweat, hair, and breast milk). The average concentration of arsenic in sweat induced in a hot and humid environment was 1.5 µg/L, and the hourly loss was 2 µg (Vellar 1969). With an average arsenic concentration in the skin of 0.18 mg/kg, Molin and Wester (1976) estimated that the daily loss of arsenic through desquamation was 0.1-0.2 µg in males with no known exposure to arsenic. As mentioned above, the excretion of arsenic in breast milk is low.
A physiologically based pharmacokinetic (PB-PK) model for exposure to inorganic arsenic (orally, intravenously, and intratracheally) in hamsters and rabbits has been developed (Mann et al. 1996a). It consists of five tissue compartments (i.e., liver, kidney, lungs, skin, and other organs) and takes into consideration the absorption, distribution, metabolism, and excretion of arsenate, arsenite, MMA, and DMA; the four major metabolites of inorganic arsenic. The model was found to simulate accurately the excretion of arsenic metabolites in urine and feces.
The model has been extended to a PB-PK model for humans by taking into consideration species differences in absorption and metabolic rate constants (Mann et al. 1996b). It describes the absorption and distribution of arsenic following oral intake or inhalation of As(III), As(V), or both. The extended model was validated against empirical data on the urinary excretion of the different metabolites of inorganic arsenic following repeated oral intake of arsenite, intake of inorganic arsenic via drinking water, and occupational exposure to arsenic trioxide. Predicted variation in urinary excretion of arsenic metabolites in relationship to form of arsenic absorbed, route of absorption, and time of urine sampling needs to be validated further. The model predicted a slight decrease in the percentage of DMA in urine with increasing single-dose exposure (highest dose of arsenic at 15 µg/kg of body weight), especially following exposure to As(III), and an almost corresponding increase in the percentage of MMA. A decrease in the percentage of DMA of about 10% corresponded to an increase in the arsenic dose of about 1,000 µg.
One major objective of the model was to compare the urinary excretion of arsenic metabolites under different exposure conditions. Examples showed that consumption of drinking water containing arsenic at 50 µg/L by adults results in a higher urinary excretion of arsenic than that following occupational exposure at 10 µg/m3 (Mann et al. 1996b).
A PB-PK model for ingested arsenate in mice (Menzel et al. 1994; Menzel 1997) assumed efficient absorption of arsenate in the gastrointestinal tract and metabolism of arsenic mainly in the liver. Because the tissue concentrations of As(III) could not be explained by absorption of As(III) from the blood (the concentration in blood was very low), all organs were assumed to have some capacity to metabolize As(V) to DMA. Tissue binding was mainly in the form of As(III), and the binding constant was calculated from experimental data. The model involved complex elimination from the kidneys.
Summary And Conclusions
When ingested in dissolved form, inorganic arsenic is readily absorbed in the gastrointestinal tract. Arsenic appears to be poorly absorbed through intact human skin but can bind externally to skin and hair. Absorbed arsenic is transported in the blood, bound to SH groups in proteins and low-molecular-weight compounds such as GSH or cysteine, to the organs in the body. Studies on blood arsenic concentrations in hemodialysis patients indicate that part of arsenic is bound to transferrin. The extent of the binding in healthy individuals is not known. Most of the arsenic in blood is cleared
with a half-time of about 1 hr. The whole-body half-time of ingested arsenite is about 4 days, urine being the major excretory pathway.
In humans, inorganic arsenic is methylated to MMA(V) and DMA(V), which are less reactive with tissue constituents, less acutely toxic, and more readily excreted in the urine than inorganic arsenic. The methylation involves addition of methyl groups from S-adenosylmethionine to arsenic in its trivalent oxidation state. A major part of absorbed pentavalent arsenic is reduced probably by GSH or cysteine. Thus, the tissue distribution, retention and toxicity of arsenic following exposure to moderate doses of arsenite and arsenate are similar. At very high doses, more arsenic is retained following exposure to arsenite than to arsenate. The liver is an important initial site of arsenic methylation, but most tissues seem to have methylating capacity. There are major differences in the biotransformation of inorganic arsenic between animal species and population groups. Most experimental animals methylate arsenic more efficiently and excrete less MMA in the urine than do humans. Some mammals (e.g., chimpanzee, marmoset monkey, and guinea pig) have been identified that do not methylate inorganic arsenic at all. Although the rat efficiently methylates arsenic, a major part of the DMA produced is retained in the erythrocytes. That response and the unusual biliary excretion of arsenic in the rat make it a less-suitable animal model for studies of arsenic disposition in humans.
In people occupationally, experimentally, or environmentally exposed to inorganic arsenic, the urinary content of metabolites of inorganic arsenic generally consists of 10-30% inorganic arsenic, 10-20% MMA, and 55-75% DMA. Some groups of people who excrete only a few percent of MMA have been identified. That response, together with marked individual variations, can indicate a genetic polymorphism in the arsenic methyltransferases. Experimental studies indicate that the methylation of arsenic might also be influenced by the arsenic species absorbed, by acute high-level exposures, as well as by nutritional factors and diseases.
Animal studies have shown retention of arsenic in the skin, hair, squamous epithelium of the upper gastrointestinal tract, epididymis, thyroid, skeleton, and lens of the eye. Arsenite is the main form interacting with tissue constituents, except the skeleton. In human subjects, the hair and nails have the highest concentrations of arsenic (0.02-1 mg/kg of dry weight), and the skin and lungs have fairly high concentrations (0.01-1 mg/kg of dry weight). Data extrapolated from animal studies permit the development and validation of a suitable PB-PK model for inorganic arsenic for humans.
Experimental animal studies show that both inorganic arsenic and the methylated metabolites pass the placenta. In humans exposed to arsenic via drinking water, arsenic concentration in cord blood was similar to that in
maternal blood. DMA was the main form of arsenic in plasma of mothers and newborns. The excretion of arsenic in human milk is low, and in areas with high arsenic concentrations in the water, an infant is less exposed to arsenic via breast feeding than via formula prepared from the water.
Because of interspecies differences in the amounts of various arsenic species excreted in the urine and the amounts of methyltransferases in tissues, extrapolation of animal data to humans is generally not possible. More human studies are needed, including research using human tissues, to answer some of the questions concerning the disposition and toxic effects of arsenic. Factors influencing methylation, tissue retention, and excretion of arsenic in humans (e.g., arsenic-binding proteins) also need to be investigated.
Other studies of less critical importance but nonetheless needed to fill important data gaps include the following:
Studies to determine whether the methylation of arsenic in vivo results in the formation of reactive intermediates that are distributed to tissues.
Studies to identify the gene or genes for arsenic methyltransferases so that nucleotide probes can be used to examine the relationships between the polymorphism of arsenic methyltransferases and the phenotype (excretion of arsenic metabolites in urine), as well as between the polymorphism of arsenic methyltransferases and the signs and symptoms of arsenic toxicity.
Studies on the bioavailability of inorganic arsenic in various types of food.
Studies to examine fetal exposure to various arsenic metabolites during different stages of development.
Studies using arsenic methyltransferase knock-out mice to determine whether methylation alters inorganic arsenite toxicology.
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