Mercury Exposure and Antinuclear Antibodies Among Females of Reproductive Age in the United States: NHANES

Emily C. Somers; Martha A. Ganser; Jeffrey S. Warren; Niladri Basu; Lu Wang; Suzanna M. Zick; Sung Kyun Park

Disclosures

Environ Health Perspect. 2015;123(8):792-798. 

In This Article

Discussion

In this population-based study, we found that mercury exposure was associated with increased risk of high-titer ANA positivity among reproductive-age females in the general U.S. population. Specifically, this association appears to be driven by organic (methyl) mercury, the predominant species in hair and total blood. Notably, a dose–response relationship was observed for low methylmercury exposure levels (< 0.37 ppm hair mercury; < 1 μg/L total blood mercury), in the range generally considered safe for women of childbearing potential by regulatory agencies (Mergler et al. 2007). The predominant nuclear staining pattern of speckled found in our population is a marker of autoimmunity with a wide variety of clinical associations, including SLE, mixed connective tissue disease, Sjögren's syndrome, and idiopathic inflammatory myopathies (Morehead 2008). The methylmercury association was robust across models, whereas other suspected risk factors in the multivariable models, including age and smoking, were not associated with ANA risk.

Our findings are compatible with murine data demonstrating development of autoimmunity in response to methylmercury exposure in genetically susceptible strains (Häggqvist et al. 2005; Hultman and Hansson-Georgiadis 1999). Results from human studies have been inconsistent regarding the relationship between organic mercury and autoimmunity. An ecologic study of two Brazilian riverine communities with high fish consumption found a suggestion of higher ANA prevalence in the community with higher average hair mercury levels (8 ppm vs. 6.4 ppm) (Silva et al. 2004). A further study in a riverine Brazilian community failed to detect an association (Alves et al. 2006); the mean hair mercury level in that study was 34.5 ppm, thus it is possible that a dose effect between methylmercury and ANA positivity could have been obscured if, as our data suggest, the response plateaued at a low exposure threshold. A study of females 12–85 years of age from one cycle of NHANES (2003–2004) failed to detect a significant association between total blood mercury and ANA positivity, although the nonlinear nature of association that we observed was not addressed in their analyses (Gallagher et al. 2013). Further, they did not report the titer for defining ANA positivity, and in their smaller sample (632 compared with 1,352 in our blood mercury analyses) statistical power may have been inadequate to detect an association.

Our study focused on females 16–49 years of age. It is well recognized that females have a higher risk of autoimmune diseases (Cooper et al. 2009; Somers et al. 2007, 2013, 2014), and that risk among females may also correlate with reproductive stage. Moreover, estrogenic hormones may promote autoimmunity (Somers and Richardson 2014). Mercury metabolism may also contrast between sexes, and differences in mercury excretion and distribution have been observed between sexes in mouse models (Hirayama and Yasutake 1986; Hirayama et al. 1987), as well as immunotoxic effects at lower internal doses in females (Nielsen and Hultman 2002).

Oxidative stress has been shown to contribute to the induction of autoimmune phenotypes in animal models, such as through epigenetic mechanisms converting normal helper T cells to autoreactive lymphocytes sufficient to cause lupus in the absence of added antigen (Somers and Richardson 2014). Mercury induces oxidative stress through sulfydryl reactivity and depletion of cellular antioxidants (Ercal et al. 2001). In human T cells treated with methylmercury, reductions in intracellular glutathione (GSH) concentration, glutathione S-transferase activity, and mitochondrial transmembrane potential have been observed, followed by generation of reactive oxygen species; intracellular GSH depletion has further been linked to susceptibility of T cells to undergo methylmercury-induced apoptosis (Shenker et al. 1999).

It is unclear why we did not find evidence linking inorganic mercury to ANAs because inorganic mercury has been more thoroughly linked to autoimmunity in animal models (Vas and Monestier 2008) and industrially exposed human populations (Cooper et al. 2004; Gardner et al. 2010; Silva et al. 2004). However, the higher doses in such studies limit their generalizability. Indeed, median urinary mercury levels were > 3.7 μg/L in a pair of studies in a Brazilian gold-mining population (Gardner et al. 2010; Silva et al. 2004) (compared with our median of 0.64 μg/L). Another distinction is that these studies used an ANA titer of ≥ 1:10 as detectable as well as a restricted dilution range (to 1:320); a more robust approach would be to employ higher titration levels to better assess strength of ANA positivity. Mechanisms of and degree of immunotoxicity may differ according to level of inorganic mercury. For instance, mercuric chloride at high concentrations (40 μM) has been associated with nonapoptotic cell death, rapid cellular permeabilization (Pollard et al. 1997), and modification of the nucleolar antigen fibrillarin from a 34-kDa non-disulfide–bonded form to a 32-kDa disulfide–bonded form. It is conceivable that structurally altered fibrillarin would be more immunogenic than its native form by unveiling of cryptic epitope(s), and together with cellular necrosis and permeabilization, could be more readily accessible to the immune system. At lower concentrations, fibrillarin migrated at both 32-kDa and the predominant 34-kDa forms, and greater cellular viability was maintained (Pollard et al. 1997). In contrast to inorganic species, organic mercury is lipophilic and more readily crosses cellular membranes, but it may demethylate intracellularly to inorganic mercury (Clarkson and Magos 2006), which may ultimately be more immunotoxic. It is plausible that subcytotoxic levels of organic mercury, such as those in our study, over long periods might yield higher intracellular doses of inorganic mercury and more efficient access to the nuclear environment than would occur with direct exposure to similarly low levels of inorganic mercury.

In our study, we found that speckled patterns predominated (96% of ANA positives). A variety of speckled ANA patterns can be seen by indirect immunofluorescence. Antigen specificities include U1-SnRNP (small nuclear ribonucleoproteins), Sm (Smith), U2-snRNP, U4/U6-snRNP, SSA/Ro, SSB/La, and other less common antigens (Bradwell et al. 2003). In contrast, the nucleolar pattern has primarily been reported in association with inorganic and methylmercury, with specificity of autoantibody formation to fibrillarin/U3RNP demonstrated in murine models in response to mercuric chloride (Hultman et al. 1989; Reuter et al. 1989). A proposed mechanism is that inorganic mercury cross-links with free sulfhydryls on two cysteines of fibrillarin, resulting in physiochemical protein modification (Pollard et al. 1997). Although anti-fibrillarin antibody formation is best recognized in response to inorganic mercury, anti-chromatin and anti-histone antibody formation have also been demonstrated (Hultman et al. 1996). For all three types of autoantibodies, the response varies according to mouse strain, underscoring the relevance of genetic susceptibility. Hultman et al. (1996) demonstrated that antibodies to fibrillarin and chromatin tended to persist several months following cessation of mercuric chloride treatment, whereas anti-histone antibodies resolved more rapidly. We found only 14 cases with the nucleolar pattern, none of which demonstrated anti-fibrillarin antigenicity upon immunoprecipitation. Only 3 cases had a nuclear homogeneous pattern, which would be compatible with histone or chromatin antigens. In humans, the nucleolar pattern, and particularly anti-fibrillarin antibodies, are associated with scleroderma, especially among blacks and males (Arnett et al. 1996). The rarity of scleroderma (prevalence ~ 27.6/100,000 adults) (Mayes et al. 2003) and its associated autoantibodies make it unlikely that our study would have adequate power to detect an association with these specific autoantibodies. However, it is difficult to explain why the speckled pattern was prominent in our study but not in the animal literature or human occupational studies. Whether organic mercury preferentially targets different nuclear antigens than does inorganic mercury, or whether an alternate biologic pathway is relevant to low compared with high doses of either species, remains to be elucidated.

Fish consumption is an exposure route common to both methylmercury and essential nutrients that may have beneficial impact on the immune system. Both organic and inorganic mercury are suggested to increase the production of prostaglandin E2 and phospholipase A2 (Mazerik et al. 2007), leading to release of arachidonic acid. Omega-3 fatty acids are an alternative substrate to arachidonic acid for cyclooxygenase and lipoxygenase enzymes, and they induce a series of anti-inflammatory eicosanoids (Simopoulos 2002). Thus, we incorporated omega-3 fatty acids into our modeling because of the potential for negative confounding (Choi et al. 2008). There was indeed a modest increase in the magnitudes of association for hair mercury when adjusting for omega-3 intake. PCBs are persistent toxicants with suggested immune effects (Gallagher et al. 2013; Heilmann et al. 2010) and fish consumption as an exposure route. The hair and total blood mercury associations were not appreciably altered with inclusion of PCBs in the models.

Limitations of our study include its cross-sectional nature, precluding the ability to determine the pattern and chronicity of mercury exposure, and persistence of ANA positivity or future risk of overt disease. However, the study of risk factors for preclinical disease is an important tool for dissecting the etiology of complex diseases with long latencies (Cooper 2009). Further, certain combinations of autoimmune diseases tend to co-occur within individuals and families (Cooper et al. 2009; Somers et al. 2006, 2009) to an extent inadequately explained by genetic background. The identification of shared environmental factors for immune dysregulation relevant to a variety of autoimmune phenotypes is an important goal (Dietert et al. 2010). The nonspecific nature of the ANA patterns documented here supports the premise of organic mercury as a risk factor for multiple autoimmune conditions. Future research is necessary to evaluate whether our study findings extend to other populations, including males and persons outside of the 16- to 49-year-age range.

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