Though some of the genes and pathways are similar, the major finding of the study is that almost all of the genes that correlate with circulating Hg levels in AU boys are different from TD boys, and almost all of the genes that correlate with circulating Hg levels in TD boys are different from AU boys. These transcriptional differences were observed even though the Hg levels were low and not significantly different between the groups in our study. Since the data reported are only correlations, no conclusions can be drawn as to whether Hg plays any role in the pathogenesis of autism based upon the current results. Given the low Hg levels and the fact that this is a cross-sectional study, no cause and effect relationship should be drawn from the current data. Though the data can be interpreted in several ways, we suggest that the different transcriptional programs associated with Hg in AU compared to TD subjects may be related to the genetic differences in the two groups of children.
Common Genes (List A and List B) and Common Pathways
The major finding of this study is that very few genes that correlated with Hg levels in AU subjects also correlated with Hg levels in TD subjects. There were only 15 genes that correlated with Hg levels in both the TD and AU groups—and that were not significantly different between the two groups. These genes were involved in apoptosis, the immune response, and response to oxidative stress. Moreover, there were only 11 genes whose expression correlated inversely with Hg levels in AU compared to TD subjects—that is genes correlating with Hg levels in both groups but in opposite directions. These genes were involved in pathways contributing to neuronal development and neuronal survival and cell death. Notably, one gene (FLNB, filamin B, beta (actin binding protein 278)), which was positively correlated with Hg levels in TD and negatively correlated in AU subjects, is involved in anchoring and biogenesis of cytoskeleton and organization of actin filaments (Xu et al.
1998; Tu et al.
2003; Leedman et al.
1993). A number of studies show that most heavy metals, including zinc, arsenic, mercury, and cadmium, can disrupt actin and microtubules in intact cells (Templeton
2000).
The analysis of all genes correlating with Hg levels in all AU and TD subjects showed a number of common networks. Figure
8 is a good example where different genes from the different groups are regulated within the same network. This could suggest that similar functions might be accomplished with different genes in AU compared to TD, and that homeostasis is maintained in spite of the genetic differences of AU and TD subjects.
Genes Correlating with Hg Levels in TD but not in AU Boys (List C)
Thus, most of the genes whose expression correlated with Hg levels in the TD group were different from those whose expression correlated with Hg levels in the AU group. For the TD subjects, the top biological function for the genes that correlated with Hg levels was cell death. Some of these genes were involved in apoptosis of neurons (NFATC4, BAD, BDNF) (Graef et al.
2003; Ottilie et al.
1997; Hoey et al.
1995; Botta et al.
2004; Kerschensteiner et al.
1999; Mamounas et al.
1995), survival of neurons, recovering and regeneration of axons (BDNF, RTN4) (Kerschensteiner et al.
1999; GrandPre et al.
2000), apoptosis of pre-T lymphocytes (BCL2L11) (Bouillet et al.
1999), survival of pro-B lymphocytes, and survival of memory B cells (BRAF, BCL2L11, BDNF, TCF3) (Oliver et al.
2004; Fischer et al.
2007). It is interesting that most of these correlations are negative. Chronic exposure to low concentrations of heavy metals, including mercury, causes immune dysfunction related in large part to apoptosis of leukocytes (Pollard and Hultman
1997; Zelikoff and Gardner
1996; Laiosa et al.
2007; Madureira et al.
2007; Ziemba et al.
2005; Mondal et al.
2005; McCabe et al.
2003,
2005; Cunha et al.
2004; Sarmento et al.
2004; Kuo and Lin-Shiau
2004; Colombo et al.
2004; Field et al.
2003). Both inorganic and organic mercurials cause human T-cell apoptosis with mitochondria being the target organelle for the induction of cell death (Shenker et al.
1999). In addition, microarray analysis of Hg-induced changes in gene expression in human liver carcinoma (HepG2) cells revealed genes involved in apoptosis and metabolic regulation with significant effects on genes involved in immune system pathways (Ayensu and Tchounwou
2006). However, the levels of Hg applied to the cells were much higher than the Hg levels of the subjects in our study.
The second major biological function for genes that correlated with Hg levels in TD subjects was cell morphology and cell death. Mercury-induced cell death can be apoptotic or nonapoptotic. The nonapoptotic cell death produced by mercury is associated with a specific cellular morphology (Pollard et al.
1997,
2000). Mercury-induced apoptosis results from dys-regulated signal transduction that leads to inactivation of enzymes, generation of free radicals and peroxides, protein conformation changes, and inhibition of transport processes which disrupt cell membrane permeability (Tchounwou et al.
2003; Clarkson and Magos
2006; Counter and Buchanan
2004; Zahir et al.
2005). Genes associated with transmembrane potential and permeabilization of mitochondria correlated with Hg levels in TD subjects in our study. Mercury-induced membrane changes of mitochondria associated with glutathione imbalance and antioxidant defenses have been reported in TD versus AU transformed lymphoblastoid lines (James et al.
2009). Mercury increases mitochondrial permeability, which is characterized both by a decline in transmembrane potential and intracellular pH, as well as the generation of reactive oxygen species and a decline in T-cell GSH content (Shenker et al.
2000). In our study, Granulysin (GNLY) showed a positive correlation with Hg levels in the TD group. GNLY is a cytolytic and pro-inflammatory molecule, which contributes to mitochondrial damage (Krensky and Clayberger
2009). B-cell lymphoma 2-like protein 11 (apoptosis facilitator) (BCL2L11) showed a negative correlation with Hg in the TD group in our study. BCL2L11 (also known as BIM) is a BH3-only protein from the Bcl-2 family (Youle and Strasser
2008), whose family members are the main regulators of programmed cell death via the mitochondrial (intrinsic) apoptotic pathway. Interactions between pro- and anti-apoptotic proteins of the Bcl-2 family decide the fate of cells in response to stress signals. BCL2L11 is essential for hematopoietic homeostasis and thymocyte negative selection, and suppresses autoimmunity (Youle and Strasser
2008).
Some of the cell morphology genes that correlated with Hg levels in TD subjects also have functions within the central nervous system. These included genes that affect neuronal survival (ABL1 (+), BDNF (−)), sprouting and projection of axons (BDNF (−), RTN4 (−)), plasticity of the synapse (BDNF (−), NFATC4 (+), PMCH (−)), and remodeling of axons (SFRP1 (−)) (Kerschensteiner et al.
1999; GrandPre et al.
2000; Gascon et al.
2007; DeFreitas et al.
2001; Mamounas et al.
2000; Fournier et al.
2001; Seil
1998; Groth and Mermelstein
2003; Krylova et al.
2002). It is possible that mercury affects these genes in the brain as well as the blood, and this might relate to neurotoxicity observed following exposure to much higher levels of mercury (Rice
2008; Guzzi and La Porta
2008).
We observed a negative correlation between GCLM and Hg levels in TD, but no correlation in AU. GCLM is a member of the glutamate-cysteine ligase complex, which is the first rate-limiting enzyme of the glutathione synthesis, and is the major route of mercury detoxification.
James et al. (
2009) observed a reduced glutathione reserve capacity in both cytosol and mitochondria in lymphoblastoid cells derived from children with autism when compared to children without autism. Glutathione reserve capacity is an indicator of systemic redox status that can be used to assess and treat individuals at risk of oxidative stress-related pathology (Jones
2006a,
b). In our study, we observed a negative partial correlation between GCLM transcript abundance and Hg levels in the TD group, while there was no significant correlation in the AU group. GCLM is transcriptionally regulated in human HepG2 cells (Lee et al.
2006). We also observed a negative partial correlation between GGT1 transcript abundance and Hg levels in the AU group, while there was no significant correlation in the TD group (see section “Genes Whose Expression Correlates with Hg Levels in AU but not in TD Boys (List D)”). GGT1 is involved in the utilization of
l-glutamate,
l-cysteine, and glutathione metabolism. It is involved in the generation of
l-glutamate, which is a substrate for glutathione synthesis. Thus, if there is negative regulation of this gene, there may be less substrate for the glutathione biosynthesis pathway, which may affect the redox status. Overall, our studies of transcript abundance in blood of AU and TD boys are difficult to relate to the in vitro studies of AU and TD lymphoblastoid cell lines except to say that both approaches show associations between genes in the glutathione pathways and Hg levels.
CDKN2AIPNL and ZMYM6 negatively correlated with Hg levels in this study and both were downregulated with mercury treatment in human liver carcinoma (HepG
2) cells (Ayensu and Tchounwou
2006). ALDH3B1, RPS9, SUB1, and VPS25 also were negatively correlated with Hg in TD subjects in this study and were downregulated in Hg-treated yeast (Jin et al.
2008).
Genes whose expression correlated similarly with Hg levels both in TD and AU (List A) and whose expression correlated with Hg levels only in the AU group (List D) have transcription factor (TF) binding sites for SP1 and Kruppel-like factor (KLF)-like TFs, respectively. KLF6, which is an Sp1-like zinc finger transcription factor, is a transactivator of inducible nitric oxide synthase (iNOS) (Warke et al.
2003), which produces NO and modulates inflammatory signals and oxidative stress (Mungrue et al.
2003).
Genes Whose Expression Correlates with Hg Levels in AU but not in TD Boys (List D)
One of the top biological functions in the list of genes that correlated with Hg levels in AU but not TD boys was amino acid metabolism. The genes included gamma-glutamyltransferase (GGT1/2/3; probe 244179_x_at); as well as solute carrier family 25 (mitochondrial carrier: glutamate), member 22 (SLC25A22 (−)), and solute carrier family 7 (cationic amino acid transporter, y+ system), member 8 (SLC7A8 (−)) which are involved in the transport of
l-amino acids (Pineda et al.
1999). SLC7A8, which had negative partial correlation with Hg levels in the AU group in our study, was upregulated in mercury-treated yeast cells (Jin et al.
2008).
FAF1 (Fas-associated factor 1) expression was positively correlated with Hg levels in AU but not TD subjects. Fas is a member of the tumor necrosis receptor factor family, which can induce apoptosis when activated by Fas-ligand binding (Chu et al.
1995). Apoptosis induced by Fas plays an important role in the development and function of the immune system (Lowin et al.
1994; Nagata
1994). Genetic studies in mice show that defects in Fas-mediated apoptosis resulted in abnormal development and function in the immune system (Takahashi et al.
1994). FAF-1 also inhibits the NF-κB activation induced by various stimuli, including tumor necrosis factor-α, interleukin-1β, and lipopolysaccharide (Park et al.
2004). NF-κB is a central regulator of genes involved in the immune response, inflammatory response, and apoptosis (Beg and Baltimore
1996; Pahl
1999; Baldwin
1996).
CHCHD4 (negatively correlated with Hg level in AU), ELOVL1, SLC7A8, and Cxorf34 (positively correlated with Hg level in AU) responded to mercury in yeast in the same direction as in our AU subjects. NTHL1 (positively correlated with Hg level in AU) was downregulated in yeast after mercury treatment, whereas MYBBP1A (positively correlated in AU) was up- or downregulated depending on the treatment dose (Jin et al.
2008).
HLA-DQ, an αβ heterodimer of the MHC Class II type which is a cell surface molecule found on antigen presenting cells, presents antigens to T-lymphocytes (Germain and Margulies
1993; Janeway et al.
1997; Kwok and Nepom
1998). The α and β chains are encoded by HLA-DQA1 and HLA-DQB1, respectively, and both the α- and β-chains vary greatly in the population. Different DQ isoforms can bind to and present different antigens to T-cells. Several lines of evidence suggest that mercury toxicity depends on the mercury species, dosage, individuality, and the type of MHC association (Ayensu et al.
2004; Hanley et al.
1997,
2002). Moreover, chronic exposure to low concentrations of heavy metals such as mercury results in immune dysfunction (Ayensu et al.
2004; Hanley et al.
1997,
2002). Immune dysregulation has been observed in autism, and has been postulated to play a role in the development and pathogenesis of autism in some cases (Warren et al.
1996,
1997; Singh and Rivas
2004; Ashwood et al.
2006; Gupta et al.
1996). In this study, we found that HLA-DQA1 was negatively correlated, whereas HLA-DQB1 was positively correlated with Hg levels in AU but not TD subjects. They both were downregulated in response to mercury in human liver carcinoma (HepG
2) cells (Ayensu and Tchounwou
2006).
Hg Levels in AU and TD
Hg levels in children from the CHARGE study with and without autism have been studied in detail (Hertz-Picciotto et al.
2009). The Hg levels in the TD and AU groups were quite similar in this sample to those in the larger CHARGE sample: means of 0.60 μg/l for TDs in both groups, and means of 0.46 and 0.49 μg/l for the AU/ASD children in this subsample and the larger study (Hertz-Picciotto et al.
2009). Thus, the sample of subjects in this study is representative of the CHARGE study as a whole. None of the samples used in this study had Hg levels higher than the EPA reference level of 5.8 μg/l. The median Hg concentration and inter-quartile range were comparable to those of the U.S. population of children ages 1–5 years in 1999–2002 (McDowell et al.
2004). It is notable, therefore, that the gene expression differences described here occurred in the EPA allowable range. This is consistent with previously reported dose-dependent effects of Hg (Tchounwou et al.
2003; Clarkson and Magos
2006; Counter and Buchanan
2004; Zahir et al.
2005) and with a dose-dependent transcriptional response in human liver carcinoma cell lines treated with mercury (Ayensu and Tchounwou
2006). It is important to emphasize that acute intoxication with much higher mercury concentrations or even chronic exposure to much higher Hg concentrations might produce different transcript associations.