The work described here introduces a new transgenic model in which to study the effects of iAs in vivo. Although it might be argued that the field of arsenic research has more than enough different experimental systems and approaches and that greater integration among existing systems is needed, we believe that the Drosophila model has some significant advantages for experimentation and more than enough homology to vertebrate cellular and molecular pathways to encourage its consideration when it comes to exploring iAs action in vivo. This is not to say that Drosophila is a perfect toxicological model because it is clear that xenobiotic metabolism, transport, and toxicokinetics are often likely to differ from that in vertebrates. However, this is an important consideration in mammalian models too and particularly so in the case of iAs toxicity where rodent models also suffer in some of these respects in comparison to the human situation. Our goal in exploiting this fly model is to aid in the elucidation of potential molecular mechanisms of iAs toxicity through manipulating the genetic background (via the agency of the system's facile classical and “reverse” genetic manipulations) and observing the consequences on various phenotypic endpoints that resemble those seen in vertebrates; these can then be tested for relevance in suitable vertebrate-based assays. Because the methylation of iAs has become an important focus of recent investigations, particularly with respect to the outcomes of chronic exposure to iAs through drinking water, the lack of an equivalent methylation system in Drosophila, far from being a drawback, actually allows numerous genetic approaches aimed at exploring mechanistic features of iAs toxicity to be envisaged. We have explored this idea by creating transgenic Drosophila that express, under inducible control, a common allele of the human AS3MT gene and have shown that the two most common MAs species found in human urine, i.e., MMA and DMA, are now produced in induced flies. Neither species is detected when the corresponding uninduced transgenic flies are fed iAs, suggesting that there is tight regulation in the system and that unique biological effects uncovered in the induced transgenic flies will be specifically attributable to the presence of MAs.
With the system thus established and characterized and using two very different phenotypic endpoints, we have gone on to show that methylated metabolites of iAs can have strongly differential dose-dependent effects. Thus, methylation can ameliorate the overtly toxic life-threatening effects of high-dose iAsIII
administration by extending life span, an effect presumably mediated by enhanced efflux of the synthesized MAs relative to iAs. Certainly, our demonstration of a lower As body burden in transgenic flies induced to methylate compared with those not induced is strongly consistent with this interpretation as being causative here. However, this same methylation of iAs significantly enhances its potential to act as a lower dose-based agent of chronic molecular damage (in this case chromosome instability), an outcome that undoubtedly contributes to pathological consequences for an organism chronically exposed to iAs over the long term. It is important to note that the doses we have used in these experiments (6–9 ppm As) are in precisely the same range as those employed in a recently reported “whole-life” exposure model of carcinogenesis developed in the mouse (Tokar et al., 2011
). Our combined observations therefore neatly integrate, in one model system, a good deal of older literature that proposed methylation of iAs was a detoxification pathway (Buchet et al., 1981
; Crecelius, 1977
) with newer data, which have suggested that the methylated species (in particular the +3 oxidized form) are, albeit indirectly, more damaging to DNA (Mass et al., 2001
) and (perhaps related to this) more active in causing cell transformation (Bredfeldt et al., 2006
) and more highly associated with disease susceptibility in human populations (Steinmaus et al., 2010
; Valenzuela et al., 2009
). It is important to emphasize that the magnitude of the phenotypic effects described in this report is only
altered when the hAS3MT
transgene is “induced” in the presence of food-borne arsenite, strongly consistent with the notion that it is the transformation of iAs to the methylated species that is solely responsible for the altered phenotypic outcomes, i.e., decreased chromosome stability under one set of dose conditions and increased life span under a different (significantly higher) set of dose conditions.
Based on the results reported here, as well as previous data on the role of MAs, we are drawn to the conclusion that such observations may best be viewed as representing two sides of a single evolutionary coin. It is reasonable to hypothesize that the various xenobiotic defense mechanisms in animals have evolved to protect the life and (most importantly from the evolutionary perspective) the reproductive capacity of the afflicted animal. Thus, it is well known that acutely toxic doses of iAs uncouple mitochondrial oxidative phosphorylation (Ter Welle and Slater, 1964
), which can rapidly lead to death. A metabolic processing system (like methylation) that leads to efficient cellular efflux of As in such a situation has the potential to preserve life and, most significantly, increase the chances of reproductive success, at least in the short term, and is therefore of distinct advantage when subjected to the forces of natural selection. There are numerous studies showing that ATP-binding cassette-type membrane transporters (e.g., multidrug resistance proteins [MRPs]) play an important role in exporting As from cells (see Thomas, 2007
), and recent data are consistent with upregulation of MRP2 protein correlating with enhanced DMA transport from hepatocytes (Drobna et al., 2010
). In other studies, methylation-competent cells accumulated less arsenic than their nonmethylating counterparts (Dopp et al., 2010
; Drobna et al., 2005
). Perhaps most important in this context are observations on an AS3MT
knockout mouse, where loss of methylation ability leads to much slower clearance of As from tissues (Drobna et al., 2009
) and consequently to an associated high accumulation of iAs (Hughes et al., 2010
) leading to rapid systemic toxicity compared with the wild-type mouse (Yokohira et al., 2010
). Interestingly, these latter studies were conducted using arsenic concentrations in the range 50–150 ppm, very similar to that (62.5 ppm) at which we tested hAS3MT-expressing and nonexpressing Drosophila
for relative viability. Thus, we see strong parallels between this work and our own studies. The fact that the same MAs species produced by normal metabolism can be detrimental to cellular macromolecules (as shown here and by others previously) will likely have no short-term consequences in terms of pathology or impaired reproductive capacity. Of course, over the long term, such exposure to MAs (as will occur subsequent to ingestion of iAs-contaminated drinking water) will cause a slow accumulation of macromolecular damage that will ultimately lead to pathological outcomes. However, such a chronically exposed animal is expected to be well beyond its prime reproductive age by the time this happens, and so the metabolic capacity to produce MAs species is most unlikely to be eliminated through the agency of natural selection. Given these considerations, it makes sense that relatively long-lived animals (such as mammals) have evolved (and retained) a system for methylating iAs that is almost certainly linked to facilitated export. Although some mammals (e.g., certain nonhuman primates) do not exhibit significant iAs methylation (due to a mutation in the relevant AS3MT
gene), we are not aware of studies on such animals that test their susceptibility to pathological outcomes after long-term exposure to iAs-contaminated drinking water, such as is encountered by many human populations. Such studies are certainly approachable with the knockout mouse. In our own model, an obviously much shorter lived animal, we will pursue such questions by developing appropriate dose-related phenotypic signatures at the cellular, subcellular, and molecular levels that are related to the presence or absence of MAs and that can be translated into an appropriate mammalian context.
The model of iAs methylation in Drosophila
developed here, in addition to revealing possible advantages and disadvantages of this metabolic process, should facilitate the pursuit of more mechanistic questions related to chronic iAs toxicity. Thus, there has been much interest in the role that polymorphic variants of the human AS3MT enzyme (in particular the Met287Thr variant) may play in individual susceptibility to the effects of long-term As ingestion (Hernandez et al., 2008
; Lindberg et al., 2007
; Valenzuela et al., 2009
). We have recently created transgenic fly lines expressing this particular variant under inducible control and plan to pursue a range of experiments under different dose regimes to determine if the Met287Thr variant can differentially affect phenotypic outcomes as compared with the “normal” human allele. Moreover, the GAL4-UAS inducible system we have built into our model offers many opportunities to vary the timing, the tissue specificity, and the degree of hAS3MT expression, all of which could be particularly informative in terms of differential toxic effects. Most importantly, we plan to exploit some of the highly refined genetic approaches available in Drosophila
(e.g., transcriptome-wide RNAi) that, in combination with our transgenic model, should lend significant insight into the molecular pathways intersected by iAs and MAs in eliciting their highly dose-dependent range of differential phenotypic effects.