Evidence suggests that Se has the capacity to ameliorate MeHg-induced effects on development in rodent models (Beyrouty and Chan 2006
; Folven et al. 2009
). Although MeHg–Se interactions have been addressed previously, the mechanisms by which Se modulates MeHg-induced neurotoxicity remain poorly characterised. The novel aspect of the present study lies in elucidating potential molecular mechanisms behind this protective effect in gestationally exposed mice using environmentally relevant chemical species of Se and MeHg and a toxicogenomic approach. The critical findings of this study were: (1) perinatal MeHg and Se (separate/concurrent) exposure induced transcriptional perturbations in murine brain; (2) MeHg, Se and MeHg in combination with Se affected key functional classes of genes related to the immune system and cell adhesion; and (3) identification of significantly differentially regulated gene clusters in response to different treatments highlighted potential mechanisms of Se amelioration of MeHg-induced toxicity.
The accumulation data showed that Se had no effect on whole tissue Hg levels. This suggests that any effect of Se was related to direct interaction with MeHg at a molecular level, rather than due to an effect related to lessening the bioavailability of MeHg for cellular uptake. However, the protocol used did not account for changes in Hg distribution at a cellular level. Further studies will be needed to test this possibility. The form of Se used in the current study may have also impacted the accumulation data. Se in SeMet is relatively slowly released (Schrauzer and Surai 2009
). This may have limited the formation of Hg–Se complexes that are known to accumulate in animal tissues (Huggins et al. 2009
Studies suggest the ratio of Hg to Se may be the critical factor in determining the toxicity of MeHg (e.g. Ralston et al. 2008
). Cerebral tissue Hg/Se molar ratios in pups subjected to the maternal MeHg diet in the present study were 1.2:1, reducing to ~0.2:1 with Se supplementation. Under such conditions, a greater ameliorative effect of Se than that observed might have been expected. The explanation for the relatively minor impact of Se may be the chemical form of Se used. One hypothesis regarding the mechanism of Se mitigation of MeHg toxicity is impairment of intracellular selenocysteine cycling (Ralston et al. 2008
). Selenocysteine is an amino acid that is specifically utilised to provide enzymatic activity in the active site of certain proteins (selenoenzymes; Hatfield et al. 2006
). Rather than being directly recycled when these proteins are catabolised, the Se component of selenocysteine is normally cleaved and reattached to cysteine later in the cycle. However, by virtue of its very strong affinity for Se (Dyrssen and Wedborg 1991
), MeHg scavenges the cleaved Se, with the result being impairment of selenoenzyme formation, and disruption of selenoenzyme function (e.g. antioxidant defence; Ralston et al. 2008
). Supplementation of Se using a poorly labile form (SeMet) in the current study, may have failed to sufficiently replace Se into the cellular selenocysteine cycle, and thus may have had limited capacity to offset MeHg effects. It has been shown, for example, that SeMet may become trapped in multiple cycles of protein synthesis as methionine equivalent, before becoming available for selenocysteine synthesis (Ralston and Raymond 2010
). However, in spite of the possible effects on selenoprotein synthesis impact on genes encoding, selenoproteins were not detected using microarray in the present study. Further studies specifically examining this important group of proteins may better discern the mechanism by which MeHg and Se interact in murine brain.
Microarray analysis provided novel insight regarding the molecular mechanisms behind Se amelioration of MeHg neurotoxicity. Although the hierarchical clustering on experimental groups revealed discrimination of treatments based on gene expression pattern, microarrays were unable to completely discriminate between individual samples assigned to different treatment groups. Consistent with other studies (Glover et al. 2009
; Padhi et al. 2008
), only a small effect of MeHg on brain gene expression was noted, suggesting that gene expression in brain is, compared with many other tissues, remarkably stable. The combination of a relatively low dose and generally low-fold changes results in global gene expression profiles, that are meaningful when averaged between biological replicates, but alone do not allow individual sample discrimination. The use of higher doses of MeHg (particularly in animals exhibiting Se depletion) would likely have generated greater effects and also more complete hierarchical clustering of samples according to treatments. However, the effects observed at higher (and unrealistic) doses of MeHg may very well be different from those that occur at lower doses.
In the current investigation, a complete validation of results between the array and qPCR was lacking. There could have been several reasons for the disagreement between results from the two methods. Most genes have several transcripts and it is possible that PCR amplicons did not always correspond to the region of probe binding (3′ bias). In the current study, if the regulation of genes in both the techniques were in the same direction, it was considered a good correspondence. The basis for this approach was that microarray is a semi-quantitative technique, while qPCR is a more sensitive methodology (Bustin 2004
); hence, it is difficult to attain similar fold changes. The reduced sensitivity of array technology may stem in part from the lasers used to scan the arrays, which had a threshold limiting the accurate measurement of gene regulation. There are no such limits with regard to qPCR, where the intensity of the fluorescence is detected by the real-time PCR machine. Spots on the array can also be saturated which could confound interpretation of microarray data. The use of different data normalisation procedures in both the techniques could also contribute towards the differences observed in gene expression values.
To explore the possibility that the relatively poor correspondence between qPCR and array data was related to biological variation (i.e. that use of distinct samples was responsible for the differences observed), qPCR data was analysed two ways. One assessment utilised the data derived only from those brains also used for array analysis, and the other included all samples. In general, the statistical analysis using these two qPCR approaches was identical, indicating that the lack of correlation was not related to inherent differences in the gene expression of individual pup brains. However, results did indicate that the response of zinc-finger genes to MeHg exposure may exhibit significant inter-individual variability.
Functional annotation enrichment analysis provided potential links between MeHg and Se exposure and identified potentially important cellular pathways of MeHg toxicity and affected genes associated with cell adhesion, immune system, stress response, and neuron communication. The effect of MeHg on the immune system has been explored in both in vitro and in vivo studies (Graevskaya et al. 2003
; Omara et al. 1997
; Thuvander et al. 1996
). A study performed on balb/c mice indicated that chronic exposure to MeHg in dams resulted in transfer of MeHg to pups which impacted thymocyte development and stimulated lymphocyte activities (Thuvander et al. 1996
). In the current study, gene clusters related to immune system were impacted by MeHg exposure.
Conversely, Se is important for the optimal functioning of the immune system and is known to regulate the expression of pro-inflammatory genes in immune cells (Vunta et al. 2008
). The appearance of several functional clusters related to the immune system under the combined exposure of MeHg and Se suggests an influence of Se on the regulation of genes involved in maintaining the immune defence of the body, possibly in order to balance the immunological effects of MeHg.
Similarly, the combined effect of MeHg and Se exposure highlighted an impact of Se on genes related to cell adhesion. Neural cell adhesion molecules (NCAMs) are cell surface glycoproteins that regulate cell–cell recognition and adhesion to guide neuronal migration, elongation and synaptogenesis (Regan and Fox 1995
). NCAMs have disulfide-bonded immunoglobulin-like segments in their extracellular domain. As such they are a likely target for MeHg toxicity (Sass et al. 2001
), and thus a putative target for Se amelioration. A study conducted with corticoid-dependent asthmatics demonstrated that Se supplementation affected the expression of adhesion molecules, which play an important role in inflammation (Jahnova et al. 2002
). The enrichment of functional clusters related to cell adhesion could be a Se-mediated mechanism to offset or repair the damage caused by MeHg. Further studies are warranted to confirm such effects since extrapolating impacts from microarray expression data, especially from murine developing brain, is complex.
Effects on both the immune system and cell adhesion were most prominent under dual Se and MeHg exposure. Functional clusters “calcium binding”, “synapse”, “cytoskeleton”, “cell differentiation”, “cell proliferation”, “transcription regulation”, and “metal binding” were also enriched under MeHg and Se co–exposure, reflecting the wide variety of genes influenced by co-administration. It is also interesting to note that the concomitant exposure regime induced the greatest overall response in gene expression change. The reason for this is unclear. A recent study by Shimada et al. (2010
) found that the concurrent exposure of MeHg with polychlorinated biphenyls also induced a much larger number of significant changes in gene expression than exposure to the individual components alone. The gene expression response to the concurrent exposure of MeHg and Se in the current investigation may reflect effects related to the strong affinity between MeHg and Se that would not be apparent when each is dosed independently.
The comparative toxicogenomic database was probed using DAVID in order to examine the relative gene expression profiles generated by exposures to toxic metals and metalloids. A functional annotation enrichment analysis was performed on genes identified to be regulated in mouse (Mus musculus) for each of four metals (MeHg, zinc, arsenic and cadmium). Functional clusters of genes related to morphogenesis/development were identified as being regulated following the exposure of each of the four metals, the only cluster found to be common to all. While other clusters overlapped between two metals, profiles were largely distinct. This is not surprising given distinct mechanisms of toxic action, and the significant variation in experimental protocols used to generate the data.
The genes Wnt3 and Sparcl1 showed Se-mediated affects on MeHg-induced changes in gene expression. Wnt3 is expressed during the development of the cerebellum and Sparcl1 is involved in cell migration (Salinas et al. 1994
; Gongidi et al. 2004
). Perturbations in these genes could hinder the development of the brain. Hence, Se may be considered a therapeutic agent in this scenario, owing to its properties in reversing MeHg-induced changes in these critical genes. The expression of Sdk2 remained unaltered by Se exposure, but was up-regulated in the MeHg treatments. Sdk2 is a mediator of homophilic adhesion and has an important role in guiding neuronal migration during development (Hayashi et al. 2005
; Yamagata et al. 2002
). The impairment of Sdk2 expression levels could have an obvious and important role in mediating MeHg toxicity. As such Sdk2 could be a potential candidate as a biomarker of MeHg exposure and/or effect. Studies on Sdk2 expression in human tissues accessible via biopsy may be warranted.
The genes Reg 1 and Tspan5 involved in differentiation and proliferation (Namikawa et al. 2005
) and neuronal differentiation (Juenger et al. 2005
), respectively, were affected by all treatments (Se, MeHg, and MeHg
Se). In the current study, the genes Mbp, Tspan5, and Sparcl1 showed changes comparable to those obtained in response to MeHg exposure in a similar study performed on rats (Padhi et al. 2008
), supporting the findings of the present study. Expression profiles of these genes in response to MeHg appear to be conserved between both rats and mice allowing interspecies extrapolation. Future studies examining the mechanisms of MeHg could focus on understanding the roles of these genes in mediating toxicity.
Another interesting feature prevalent in the gene expression data was the differential regulation of genes encoding zfp467 and zkscan16. The interactions between zinc finger proteins and MeHg toxicity have been largely overlooked in the literature. However, previous microarray data revealed a distinct effect of MeHg toxicity on the expression of mRNA encoding zinc finger proteins (Glover et al. 2009
). Both Zfp467 and zkscan16 code for zinc finger proteins that are important for RNA packaging, DNA recognition, transcriptional activation, regulation of apoptosis, protein folding and assembly and lipid binding (Laity et al. 2001
). Se alleviated the down-regulation of Zfp467 under MeHg exposure, returning expression back to control levels indicating its potential to counteract MeHg-induced damage. This suggests that these transcripts could be key targets for Se-amelioration of MeHg-induced toxicity.
A controversial finding of the present study was the lack of markers of oxidative stress identified as being differentially expressed on MeHg exposure. While this finding is consistent with previous microarray data from this laboratory (Glover et al. 2009
), it is contrary to the established dogma regarding a key mechanism of MeHg toxicity. For example, Kaur et al. (2009
) found an effect of SeMet in reducing MeHg-induced ROS production in neural cell lines. Similarly, an in vivo study found that administration of Se resulted in restoration of MeHg-induced depletion of antioxidant enzymatic activities (Agarwal and Behari 2007
). There are several possible reasons for this difference between studies. The present study sampled brains at a single time-point, which may have precluded detection of oxidative stress or ameliorating pathways. The exposure regime employed in this study (sub-chronic exposure throughout gestation), may have allowed the gradual development of antioxidant defence mechanisms, which would not necessarily be reflected in altered brain gene expression levels at the chosen sampling time. In this context, it should be noted that observed effects on neurological development in these mice were limited (Folven et al. 2009
). In contrast, the acute exposure regimes of many previous studies would be more likely to overwhelm antioxidant mechanisms, and induce significant changes in molecular pathways related to this phenomenon. This suggests that chronic perinatal exposure through a natural route may result in a significantly different toxicological profile than acute exposure studies. Furthermore, since MeHg found in seafood is bound to cysteine (Harris et al. 2003
), MeHgCys was the chemical species of mercury chosen for the present study. This is in contrast to most laboratory studies where MeHgCl is the chemical species used. In vivo studies have shown that MeHgCl and MeHgCys differ in their toxicological impacts (Glover et al. 2009
; Berntssen et al. 2004
) and this effect may also account for differences between the present study and others with respect to the impact of antioxidant stress.