Folic acid supplementation lowered total blood arsenic concentrations by increasing the methylation of InAs and MMAs to DMAs, which is rapidly excreted in urine. This was evidenced by reductions in the concentrations of InAs and MMAs in blood and an increase in the concentration of DMAs in urine.
Arsenic methylation has long been considered to be a detoxification mechanism. In the 1980s, dietary methyl donor deficiency was shown to significantly decrease total urinary arsenic excretion and to significantly increase retention of arsenic in tissues in animal models (27
); this reflects the longer half-life and greater chemical reactivity of the InAs species. Arsenicals in blood are eliminated with a 3-component exponential decay pattern. The first and quantitatively most substantial half-life for InAs is ≈1–2 d, the second is 9.5 d, and the third is 38 d; these values were determined in humans (29
). Similar patterns for InAs have been observed in rabbits and hamsters. Although the half-life of MMAs and DMAs in humans has not been determined, their initial half-life of elimination in hamsters is very short (7.4 and 5.6 h, respectively) (30
), which indicates the importance of arsenic methylation in the facilitation of elimination.
The influence of arsenic methylation on arsenic toxicity has, however, been under intense investigation in recent years. Landmark work by Styblo et al (31
) and Petrick et al (32
) in 2000 found MMAsIII
to be the most toxic metabolite, both in vitro and in vivo. DMAsIII
has been reported to have DNA-nicking activity (34
), but the extent of in vivo formation of DMAsIII
is not known. A study by Valenzuela et al (35
) indicated that DMAsIII
may represent a significant proportion of total urinary arsenic. However, the potential for artifact is high, because DMAsIII
is highly unstable and difficult to measure in aqueous solutions, and it has been found to co-elute with a sulfur-containing arsenical, potentially a breakdown product of arseno-protein compounds (36
). Moreover, DMAsIII
is very easily oxidized to DMAsV
). Data suggesting that DMAsV
is a bladder carcinogen in rats (40
) have been, to some extent, discounted in terms of human relevance because of the extraordinarly high doses employed (41
Arsenite toxicity is largely attributable to its ability to react with critical sulfhydryl groups of many enzymes. It is important that the complex of arsenic with a given protein bestows selectivity to the biological effects of arsenic (42
), and arsenic metabolites differ in their protein-binding capacity: arsenite has 3 coordination sites, MMAsIII
has 2, and DMAsIII
has only one (43
). Because a stable structure forms only when arsenic complexes with 2 sulfhydryl groups in a single protein, the stability and specificity of binding of DMAIII
with monothiols is less than that formed between InAsIII
with dithiols (42
In human populations, case-control studies indicate that persons with relatively low proportions of urinary DMAs(III+V)
and high proportions of MMAs(III+V)
are at greater risk of arsenic-related health outcomes, including skin lesions, skin and bladder cancers, and cardiovascular disease (44
). By chance, 8 of the participants in the current study had arsenic-induced skin lesions. These participants had significantly higher proportions of MMAs and lower proportions of DMAs in blood than did the participants without skin lesions (data not shown). The weight of the human evidence favors the consensus that incomplete methylation of arsenic to DMAs confers increased susceptibility to multiple adverse health outcomes.
Investigation of methylation of arsenic in human populations has been reliant until now almost entirely on the measurement of arsenic metabolites in urine, where concentrations are an order of magnitude higher than in blood and therefore are readily detectable with the use of conventional GFAA spectrometry–based methods. However, the untested, underlying assumption has been that arsenic metabolites in urine reflect arsenic metabolites in blood.
Assessment of total arsenic and arsenic metabolites in urine is complicated by several factors. First, the concentration of urine varies as a function of hydration status, which necessitates that metabolites be expressed per gram of creatinine. This expression is inherently problematic because urinary creatinine 1
) is itself a significant predictor of %InAs and %DMAs in urine (14
) is biochemically linked to one-carbon metabolism insofar as its biosynthesis consumes more methyl groups than do all other SAM-dependent methylation reactions combined (49
) differs by muscle mass and consequently by age and sex; and 4
) is influenced by other factors, such as renal function, physical activity, and diet (50
). To circumvent some of these issues, arsenic metabolites in urine often are expressed as a percentage of total urinary arsenic or as ratios, eg, the ratio of InAs to MMAs or that of MMAs to DMAs. Each of these expressions, however, limits the interpretation of the data: as one metabolite increases, another consequently decreases, and it becomes difficult if not impossible to decipher which metabolite was biologically important. The correlations between arsenic metabolites in blood and those in urine were all quite strong. Nevertheless, the problems associated with urinary analyses are considerable, and our understanding of the effects of folic acid supplementation on arsenic metabolism and elimination was greatly enhanced by the assessment of arsenic metabolites in blood.
In conclusion, folic acid supplementation of persons with marginal folate nutritional status decreases total blood arsenic by decreasing bInAs and bMMAs and increasing urinary DMAs. This finding has particularly important implications for reducing body stores of arsenic after exposure has been remediated, because adverse health outcomes are known to persist for decades after exposure has ceased (51
). Additional studies are needed, including a dose-response study of efficacy; studies to determine whether the use of L-N
5-methyltetrahydrofolate, methylcobalamin, or other agents may enhance efficacy; and studies to determine whether folic acid interventions can similarly lower blood arsenic when folate nutritional status is adequate. Large-scale long-term intervention studies will be required to determine whether folic acid supplementation can prevent arsenic-induced skin lesions or minimize cancer risks.