The long-term ingestion of arsenic-contaminated ground water represents one of the worst environmental calamities in history. Such water is extensively consumed not only in developing nations, such as Bangladesh (Parvez et al., 2006
) and parts of India (Guha Mazumder et al., 1998
), but also in more highly developed countries such as China (Yu et al., 2007
) and the United States (Steinmaus et al., 2006
). Its health effects range widely from conditions such as diabetes mellitus, peripheral vascular disease (e.g., Blackfoot disease), and neuropathy to a large variety of cancers. Interindividual genetic variations have been proposed as contributing to differences in susceptibility and response—for example, polymorphisms in either the AS3MT
(Hernandez et al., 2008
) or the GSTO
(Marnell et al., 2003
) genes have been associated with increased likelihood in developing arsenic-related diseases. Given the pleiotropic nature of disease outcomes upon arsenic exposure and the large number of pathways implicated in its biological interactions (reviewed in Kumagai and Sumi, 2007
; Rossman, 2003
), an unbiased approach to determining genes and/or pathways involved in differential susceptibility to its toxic effects at the whole organismal level seems warranted. Here, we have used natural geographic variants of D. melanogaster
as a model system to test for genetic factors present in wild-type populations that may possibly predispose to arsenic susceptibility. Historically, Drosophila
has been used to shed light on many fundamental biological processes, such as organismal development, owing to its ease of genetic manipulation and analysis. However, it has become an increasingly used model system to study human disease processes and signal transduction, owing to its unexpectedly high content of cognates to many genes involved in human genetic disease and metabolic pathways (see Introduction).
By employing a combination of classical chromosomal segregation and microsatellite marker-based recombination analyses, together with the creation of a series of overlapping deficiencies in the inferred region of interest, we identified a small region of the X chromosome (defined by Df #11 and encompassing cytological location 16F1) as being of particular interest with regard to arsenic susceptibility. The genomic sequence information available in FlyBase for this region, together with the inferred annotation of its genetic function, shows that a direct sequence duplication has apparently occurred, such that two copies of the GS gene are present, along with two copies of an adjacent gene encoding a cell adhesion protein and a single gene encoding a protein with carbonate dehydratase activity.
Given well-documented observations that GSH appears to play a role in the defense of cells against arsenic toxicity (Brambila et al., 2002
), it was obviously of interest that an enzyme involved in the biosynthesis of GSH was implicated by our genetic analysis. On the other hand, that this enzyme might be GS seemed somewhat surprising in light of the prevailing view that it is the enzyme that precedes GS in the two-step GSH biosynthetic pathway, namely GCL, that provides the rate-limiting step (reviewed in Griffith and Mulcahy, 1999
). Indeed, when we examined a fly line with a P-element insert in the 5′ UTR of the Gclm
gene (encoding the regulatory subunit of the heterodimeric GCL enzyme) that causes about twofold reduction in GSH levels, it displayed very high sensitivity to arsenite, confirming previous studies performed in Gclm
knockout mouse embryo fibroblasts (Kann et al., 2005
). Thus, one prediction would be that moderately reduced GS expression, as anticipated in Df #11 owing to its 50% reduction in GS
gene dose, would affect neither overall GSH levels in the cell nor its arsenite sensitivity, as long as its substrate (γ-glutamyl cysteine derived from the GCL-catalyzed step) was present at normal levels. The first part of this prediction was true, since we found that GSH levels in Df line #11 appeared similar to those of its nondeficient parent (data not shown). However, such deficiency flies showed distinct arsenite sensitivity and encouraged us to investigate the role of GS in arsenite sensitivity in greater detail.
RNAi-based knockdown analysis in S2 tissue culture cells amply confirmed that reduction in the expression of GS
produced sensitivity to arsenite. Most strikingly, however, whole-organism knockdown of GS
(to approximately 30% of normal levels) induced extreme sensitivity, with complete developmental toxicity occurring at up to 10-fold lower concentrations (and potentially even less) than those at which the first conspicuous effects on adult eclosion typically start to occur. Particularly noticeable was the fact that this toxicity appeared to occur very early in development (presumably shortly after embryo hatching), since very few active larvae could be observed under these conditions. Though these data seemed highly contradictory based on the prediction outlined above (in support of which Drosophila
GCL has been shown to be rate limiting, Fraser et al., 2002
, as in other organisms), they make a good deal more sense when the pathway for GSH biosynthesis is viewed in a broader context. The key to this is understanding that GSH is not a static component in the cell under conditions of arsenite-induced stress. This is because it is actively bound by arsenite in a stable As(GSH)3
complex (Kobayashi et al., 2005
), which not only ties up free GSH from participating in its role as an antioxidant and regulator of the redox state of the cell (many studies have shown high levels of ROS in the presence of arsenic—see Kumagai and Sumi, 2007
) but also provides the substrate for active transport of arsenic out of the cell by the multidrug resistance proteins (Leslie et al., 2004
). It is in this situation of both synthesis and active consumption of GSH that the rate-limiting properties of GCL are likely to be compromised because changes in flux through the pathway (as would be produced under arsenite stress conditions when GSH is being consumed at a much higher rate) become sensitive to other steps in both the supply and demand pathways. Furthermore, though GCL is feedback inhibited by GSH under zero or low GSH consumption conditions (forming the basis for its reported rate-limiting behavior), this inhibition will be substantially relieved in a high GSH consumption situation, allowing other control points (such as the GS-catalyzed step) to contribute to a correspondingly greater extent. Such supply and demand considerations, inherent in the biochemical approach known as metabolic control analysis, have been recently discussed in great detail, both for the GSH pathway (Mendoza-Cozatl and Moreno-Sanchez, 2006
) and for metabolic pathways in general (Moreno-Sanchez et al., 2008
). In the present case, it provides an extremely plausible rationale for why reduced GS activity sensitizes cells that are experiencing chronic stress from arsenite exposure. The fact that this effect is particularly strong when considering developmental susceptibility as compared to cultured cell susceptibility emphasizes the importance of a whole-animal model in studying mechanisms and pathways of toxicity.
These results on the effects of reduced GS expression suggested that the differential sensitivity displayed by the PVM and Oregon R 1970 strains toward arsenite might be due to sequence polymorphisms in the CG6835
genes between the two strains. Such polymorphisms could lead to differences in levels of the enzyme, differences in the levels of transcripts, differences in transcript splice variants, or differences in enzyme activity, any or all of which might then lead to differential availability of GSH under the sustained stress of arsenite intoxication. While it is clear that both strains do contain the duplicated GS
genes, we have not yet sequenced the genes and their flanking regions in the two strains in order to address these possibilities; preliminary analyses of multiple GS
transcripts (i.e., splice variants) and their levels have shown that a good deal of complexity is present (data not shown). We have also measured GSH levels in these two strains under both control and arsenite-stressed conditions and have not found obvious differences (data not shown). However, these data might easily be compromised by the fact that typical GSH assays (including the one that we used—Senft et al., 2000
) cannot distinguish between GSH and the substrate for the GS reaction (i.e., γ-glutamyl cysteine), so this distinction needs to be made in further investigations.
According to the results described here, even though the GCL heterodimer is the rate-limiting enzyme in the production of GSH under normal conditions, in the presence of arsenite (and potentially other heavy metal toxicants) optimal GS activity is required to sustain high enough levels of bioavailable GSH to protect cells, and thus an organism, against the effects of the chronically ingested toxicant. The HapMap consortium has reported single nucleotide polymorphisms in the GS
gene of individuals from different regions worldwide (The International HapMap Consortium, 2003
), and GS deficiency (whole or partial) is a well-described inherited autosomal recessive human condition (reviewed in Njalsson, 2005
). Since it is clear that the synthesis and use of GSH in defense against arsenic intoxication is a common feature of both the invertebrate model studied here and the mammalian situation, we suggest that future studies of genetic polymorphism in human populations exposed to arsenic should consider potential associations between variant alleles of genes in the GSH biosynthetic pathway (such as GCL
) and disease susceptibility.