Novel biomarkers of mercury-induced autoimmune dysfunction: a cross-sectional study in Amazonian Brazil

Abstract

Mercury is a ubiquitous environmental contaminant, causing both neurotoxicity and immunotoxicity. Given its ability to amalgamate gold, mercury is frequently used in small-scale artisanal gold mining. We have previously reported that elevated serum titers of antinuclear autoantibodies (ANA) are associated with mercury exposures of miners in gold mining. The goal of this project was to identify novel serum biomarkers of mercury-induced immunotoxicity and autoimmune dysregulation. We conducted an analysis of serum samples from a cross-sectional epidemiological study on miners working in Amazonian Brazil. In proteomic screening analyses, samples were stratified based on mercury concentrations and ANA titer and a subset of serum samples (N=12) were profiled using Immune Response Biomarker Profiling ProtoArray protein microarray for elevated autoantibodies. Of the up-regulated autoantibodies in the mercury-exposed cohort, potential target autoantibodies were selected based on relevance to pro-inflammatory and macrophage activation pathways. ELISAs were developed to test the entire sample cohort (N=371) for serum titers to the highest of these autoantibodies (anti-glutathione S-transferase alpha, GSTA1) identified in the high mercury/high ANA group. We found positive associations between elevated mercury exposure and up-regulated serum titers of 3760 autoantibodies as identified by ProtoArray. Autoantibodies identified as potential novel biomarkers of mercury-induced immunotoxicity include antibodies to the following proteins: GSTA1, tumor necrosis factor ligand superfamily member 13, linker for activation of T cells, signal peptide peptidase like 2B, stimulated by retinoic acid 13, and interferon induced transmembrane protein. ELISA analyses confirmed that mercury-exposed gold miners had significantly higher serum titers of anti-GSTA1 autoantibody [unadjusted odds ratio=89.6; 95% confidence interval: 27.2, 294.6] compared to emerald miners (referent population). Mercury exposure was associated with increased titers of several autoantibodies in serum including anti-GSTA1. These proteins play a wide variety of roles, including as antioxidants, in the regulation of pro- and anti-inflammatory cytokines, as well as danger and oxidative stress signaling. Dysregulation of these proteins and pathways is believed to play a role in autoimmune diseases such as rheumatoid arthritis, Sjögren׳s syndrome, and multiple sclerosis. Taken together, these results suggest that mercury exposure can induce complex autoimmune dysfunction and the immunotoxic effects of this dysfunction may be measured by serum titers to autoantibodies such as anti-GSTA1.

Keywords: Autoimmunity; Biomarker; Epidemiology; Mercury.

Figure 1. Differential autoantibody expression in mercury-exposed populations

Proportional Venn diagram illustrating distribution of up-regulated autoantibody serum titers (as measured by signal intensity greater than 5,000 on Immune Response Biomarker Profiling ProtoArray) as compared to internal array controls for high versus low mercury-exposed individuals (N=6 each). Overlapping area corresponds to the shared autoantibody positivity between the groups.

Figure 2. Differential autoantibody expression in mercury/ANA subgroups

Non-proportional Venn diagram illustrating distribution of up-regulated autoantibody serum titers (as measured by signal intensity greater than 5,000 on Immune Response Biomarker Profiling ProtoArray) as compared to internal array controls for high mercury/high ANA, high mercury/low ANA, low mercury/low ANA, and low mercury/high ANA groups (N=3 each). Overlapping areas correspond to the shared autoantibody positivity between groups. This is a symbolic representation of the relationships between the four groups, but these relationships are not drawn to scale. Relative proportions of the relationships between four groups are not possible in a Venn diagram (Phillips 2014).

Figure 3. Heat map of differential autoantibody levels

The baseline level to data centered about the median level of autoantibody intensities are shown for each sample within each subgroup dichotomized based on differential mercury level and ANA positivity. Fold increase in serum autoantibody levels are shown for +/+ compared to −/− average intensity for each self-protein.