Biotransformation

In general, the removal of electrons from or addition of elec- trons to the atom influences the chemical activity and therefore the ability of metallic elements to interact with tissue targets (ligands).

Examples of charge relevance in crossing lipid barriers are repre- sented by Fe²⁺/Fe³⁺ and Hg⁺/Hg⁰passages (Misra, 1992).

Among the other metabolic transformations, the most important is bioalkylation, which mercury, tin, and lead undergo in micro- organisms, whereas arsenic and selenium are additionally

bioalkylated as part of their metabolic pathways in higher organisms (Templeton, 2003).

Alkylation reactions produce more hydrophobic species, lead- ing to an increased bioavailability, penetration to cells and through the blood–brain barrier, as well as accumulation in fatty tissues.

6.4.1 Chromium

Cr^VI is rapidly reduced in vivo to Cr^V, which in turn is rapidly converted to Cr^IV and then to Cr^III. Whereas Cr^III compounds in general represent the most stable form of chromium in the environ- ment, the aromatic bidentate picolinate ligand in chromium(III) picolinate (a widely used nutritional supplement) may result in a shift of the redox potential of the complex, such that the Cr^III can be reduced to Cr^II by biological reductants (Speetjens et al., 1999).

Hepatic and, to a lesser extent, pulmonary cells and gastro- intestinal juice have some capacity to reduce in vitro Cr^VI to Cr^III (Petrilli & Deflora, 1978; USEPA, 1984).

6.4.2 Manganese

Manganese may undergo oxidation or reduction: in several enzymes, the form of manganese has been demonstrated to be Mn^III, whereas the intake of manganese was in the form Mn^II or Mn^IV. Following MnCl2 administration, manganese was detected as Mn^III and/or Mn^II complexed with proteins (Sakurai et al., 1985).

Oxidation of Mn^II to Mn^III was reported to be catalysed in vivo by caeruloplasmin and during dismutation reactions with superoxide (Archibald & Tyree, 1987). The methyl side-chain of MMT is rapidly metabolized in rat liver and lung microsomes to an alcohol, hydroxymethylcyclopentadienyl manganese tricarbonyl, and an acid, carboxycyclopentadienyl manganese tricarbonyl, by a cytochrome P-450 monooxygenase (Lynam et al., 1990).

6.4.3 Arsenic

Biotransformation of arsenic involves methylation, leading to the formation and excretion of monomethylated and dimethylated compounds. In most mammals, only trivalent arsenic species are

methylated — i.e. in the metabolism, reduction and methylation alternate (Figure 9). Possibly, As^III is bound to a dithiol, a carrier protein, before the methyl groups are attached. S-Adenosyl methionine is the main methyl donor in arsenic methylation (Vahter, 2002).

Fig. 9. Arsenic metabolism (adapted from Buchet, 2005)

Experimental studies have indicated that the liver is an important site of arsenic methylation, especially following ingestion, when the absorbed arsenic initially passes the liver. This is supported by studies showing a marked improvement in the methyl- ation of arsenic in patients with end-stage liver disease following liver transplantation (Geubel et al., 1988). However, arsenic may also be methylated in other tissues, as methylating activity has been detected in several different tissues of male mice. The highest activity was detected in the testes, followed by kidney, liver, and lung (Vahter, 2002).

Experimental findings with rat liver preparations suggest that two different enzymatic activities are involved in the methylation of inorganic arsenic in mammals (Buchet & Lauwerys, 1985). More- over, observations in humans repeatedly ingesting low inorganic

$V 2+

+2 2+

2

$V 2+

+2 +2

+2 $V 2+

2 &+

$V &+

+2 +2

$V

&+

&

+ 2+

2

$V

&+

&

+

2 + DUVHQLFDFLG

$VL9

H+ +2

DUVHQRXVDFLG

$VL,,, PHWK\ODUVRQLFDFLG 00$

H+

+2

PHWK\ODUVRQRXVDFLG

6$0H 6$++

6$++

GLPHWK\ODUVLQLFDFLG '0$

H+ +2

GLPHWK\ODUVLQRXVDFLG

6$0H6DGHQRV\O/PHWKLRQLQH

6$0H

6$+6DGHQRV\O/KRPRF\VWHLQH

arsenic doses or acutely intoxicated by As2O3 also suggest a different rate for two methylation steps and an inhibitory effect of the trivalent inorganic form for the second methylation step leading to DMA.

According to the suggested mechanism of arsenic methylation, the methyl groups react with arsenic in its trivalent form. Experi- mental studies have shown that a major part of absorbed As^V as arsenate is rapidly reduced to As^III as arsenite, probably mainly in the blood. Because arsenite is more toxic than arsenate, this initial step in the biotransformation of arsenate may be regarded as a bioactivation. However, much of the formed arsenite is distributed to the tissues, where it is methylated to MMA and DMA. It has been shown that arsenite is taken up in hepatocytes much more readily than arsenate. At physiological pH, arsenites are present mainly in undissociated form, which facilitates passage through the cellular membrane, whereas arsenate is in an ionized form (Lerman et al., 1983).

Glutathione and probably other thiols serve as reducing agents for arsenate and MMA (Buchet & Lauwerys, 1985). Depletion of hepatic glutathione in rats and hamster by buthionine sulfoximine was shown to decrease the methylation of inorganic arsenic. Arse- nate reductase activity has been detected in human liver (Radabaugh

& Aposhian, 2000).

The population variation in arsenic metabolite production indi- cates a genetic polymorphism in the regulation of enzymes respon- sible for arsenic methylation. Genetic polymorphism has been demonstrated for other human methyltransferases (Weinshilboum et al., 1999).

The role of speciation in arsenic metabolism in a case of arsine intoxication was assessed by examining the urinary arsenic species of the patient for 1 month (Apostoli, 1997; Apostoli et al., 1997). As excreted species, with quite different excretion patterns among species: arsenite excretion followed an exponential curve; an important elimination of MMA was observed early on day 1 or 2, while DMA elimination increased progressively and culminated on day 5, when MMA excretion tended to decrease. Less than 5% of the total amount was excreted as arsenate, and it disappeared after shown in Figure 10, MMA, DMA, and arsenite were the most

day 10. The conversion of arsenate to arsenite seemed to be influenced by the amount of arsenite and by synthesis of other metabolites. The fact that DMA excretion culminated after only a few days, while MMA excretion was still elevated, seems to confirm the existence of two different methylating enzymatic systems.

Arsenobetaine seemed to be excreted independently of other species, being probably linked to uptake of arsenic from meals. The amount of arsenobetaine measured in food does not seem, however, sufficient to justify the amount of arsenic metabolite measured.

Fig. 10. Excretion of arsenic species in a case of acute arsine intoxication.

As³⁺ in arsenite, As⁵⁺ in arsenate, MMA (methylarsonic acid), DMA (dimethylarsinic acid), AsB (arsenobetaine)

(adapted from Apostoli et al., 1997).

Irrespective of the type and extent of exposure, the average relative distribution of arsenic metabolites in the urine of various population groups seems to be fairly constant (e.g. 10–30% inor- ganic arsenic, 10–20% MMA, and 60–70% DMA). However, there are certain exceptions. Indigenous people living in the Andes, mainly Atacameños, excrete less MMA in urine, often only a few per cent. In contrast, people living in certain areas of Taiwan, China, seem to have an unusually high percentage of MMA in urine, 20–

30% on average. Interestingly, the Atacameños people have lived in the north of Chile and Argentina, areas with high arsenic levels in

GD\V

—JGD\

$V

$V '0$

$V%

00$

7RWDO$V

the groundwater, for thousands of years (Vahter et al., 1995; Vahter, 1999).

6.4.4 Selenium

The key reactions in selenium metabolism can be divided into three types: namely, reduction, selenoprotein synthesis, and methyl- reduced stepwise by cellular glutathione to hydrogen selenide (H2Se), and it, or a closely related species, is either incorporated into selenoproteins after being transformed to selenophosphate and selenocysteinyl transfer RNA or excreted into urine after being transformed into methylated metabolites of selenide. As a result, selenium is present mostly in the forms of covalent C–Se bonds in mammals. It is known that humans exposed to high concentrations of the element develop a garlicky breath odour characteristic of dimethyl selenide.

6.4.5 Mercury

Hydrogen peroxide catalase oxidizes elemental mercury to Hg^II in erythrocytes and tissues. Hg^II is highly reactive, readily binding to thiols. Hg⁰ oxidation converts it to a more toxic species. The oxidation of Hg⁰ takes a few minutes in blood, providing time for Hg⁰ to cross membranes (e.g. the blood–brain and placental barriers) (Halbach et al., 1988).

Intracellular Hg^II binds to metallothionein in the cytosol (Ogata et al., 1987; Liu et al., 1991); the toxicity of the metallothionein complex is less than that of Hg^II. Exposure to inorganic mercury or elemental mercury Hg⁰ induces metallothionein in the kidney, the major site of inorganic mercury deposition in the body. Hg^I is rather unstable; in the presence of sulfhydryl groups, it undergoes dis- proportionation to one atom of Hg⁰ and to one ion of Hg^II. The actions of Hg⁺ ions have been attributed to their oxidation to Hg^II species (Foulkes, 2001).

The ubiquitously distributed enzyme superoxide dismutase can catalyse Hg^II reduction to Hg⁰.

Methylmercury, in experimental animals and humans, is slowly converted to inorganic mercury in all organs, except skeletal muscle, ation (Figure 11) (Itoh & Suzuki, 1997). Inorganic selenium is

and it passes through the renal tubule as inorganic Hg^II (Clarkson et al., 1988; WHO, 1990a).

Fig. 11. The metabolic fate of selenium in the human body. Cys = cysteine;

Met = methionine; tRNA = transfer RNA; GPx = glutathione peroxidase; Sel P = selenoprotein P; DI = type 1-iodothyronine de-iodinase; TR = thioredoxin

reductase (adapted from Lobinski et al., 2000). © IUPAC

Ethylmercury compounds are more readily dealkylated than methylmercury compounds. In monkey brain, the concentration of Hg^IIincreased and the concentration of methylmercury decreased over time after methylmercury exposure (Charleston et al., 1996;

Foulkes, 2001; Magos, 2003). Thiomersal is metabolized to ethyl- mercury and then to inorganic mercury.

A small amount of methylmercury is converted to Hg⁰ in the gastrointestinal tract. The conversion of methylmercury to inorganic

6H0HW 0HW

SRRO 6HFRQWDLQLQJSURWHLQV

6HKRPRF\VWHLQH

6HF\VWDWKLRQLQH

6H&\V

+6H

&+6H+

&+6H

&+6H 6H2

6H2

XULQH

EUHDWK

XULQH

6HSKRVSKDWH

6H&\V^6H&\VW51$

6HU^6H&\VW51$

6HOHQRSURWHLQV

*3[6H,3',75 5HJXODWHGSDWKZD\

mercury may result in biliary mercury excretion. This conversion may be the rate-limiting step in methylmercury elimination. Methyl- mercury is excreted in bile as a sulfhydryl (-SH) complex, the conjugation being catalysed by glutathione transferase. These com- plexes may be reabsorbed from the gastrointestinal tract (Clarkson, 1979; WHO, 1979).

Dimethylmercury is demethylated to methylmercury within the first few days after exposure. Phenylmercury and methoxyethylmer- cury are rapidly converted to inorganic Hg²⁺ ions, since their Hg–C bonds are more readily cleaved than those in other alkylmercury