In Utero Exposure to Mercury Is Associated With Increased Susceptibility to Liver Injury and Inflammation in Childhood

Abstract

Background and aims: Nonalcoholic fatty liver disease (NAFLD) is the most prevalent cause of liver disease in children. Mercury (Hg), a ubiquitous toxic metal, has been proposed as an environmental factor contributing to toxicant-associated fatty liver disease.

Approach and results: We investigated the effect of prenatal exposure to Hg on childhood liver injury by combining epidemiological results from a multicenter mother-child cohort with complementary in vitro experiments on monocyte cells that are known to play a key role in liver immune homeostasis and NAFLD. We used data from 872 mothers and their children (median age, 8.1 years; interquartile range [IQR], 6.5-8.7) from the European Human Early-Life Exposome cohort. We measured Hg concentration in maternal blood during pregnancy (median, 2.0 μg/L; IQR, 1.1-3.6). We also assessed serum levels of alanine aminotransferase (ALT), a common screening tool for pediatric NAFLD, and plasma concentrations of inflammation-related cytokines in children. We found that prenatal Hg exposure was associated with a phenotype in children that was characterized by elevated ALT (≥22.1 U/L for females and ≥25.8 U/L for males) and increased concentrations of circulating IL-1β, IL-6, IL-8, and TNF-α. Consistently, inflammatory monocytes exposed in vitro to a physiologically relevant dose of Hg demonstrated significant up-regulation of genes encoding these four cytokines and increased concentrations of IL-8 and TNF-α in the supernatants.

Conclusions: These findings suggest that developmental exposure to Hg can contribute to inflammation and increased NAFLD risk in early life.

FIG. 1.

Summary scheme of the present investigation describing the translational research design of the study. In a mother-child cohort across six European countries, Hg blood concentration was determined from pregnant mothers, and liver enzyme and inflammatory cytokine levels were assessed in children. The in vitro experiment assessed the inflammatory effects of Hg exposure on monocyte cells, whose recruitment in the liver is known to be a major factor contributing to NAFLD. The human leukemia monocytic cell line (THP-1) was exposed to a physiologically relevant concentration of Hg (10 μM HgCl₂) for 24 hours, and then gene expression and concentration of inflammatory cytokines were assessed in RNA and supernatants, respectively. Abbreviations: Hg, mercury; HgCl₂, mercuric chloride; NAFLD, nonalcoholic fatty liver disease; PCR, polymerase chain reaction.

FIG 2.

Maternal blood Hg concentration is associated with a high-risk subgroup of children characterized by elevated ALT and increased inflammatory cytokine levels. (A) Latent variable analysis integrating serum ALT and plasma cytokine profiling in childhood with maternal blood Hg concentration during pregnancy. The thick dark green line connecting subgroup 2 and elevated ALT (defined as ≥22.1 U/L for females and ≥25.8 U/L for males) indicates that children in subgroup 2 had a higher risk for elevated ALT compared to children in subgroup 1 (reference). The dark green lines connecting the subgroups to cytokines indicate positive associations, with the width of the lines being proportional to the effect size. The thick dark green line connecting Hg to subgroup 2 indicates a positive association compared to subgroup 1. Model was adjusted for cohort, maternal age, maternal pre-pregnancy BMI, maternal education level, maternal smoking status during pregnancy, and child sex and age at outcome assessment. (B) Distribution of Hg, ALT, and inflammatory cytokines in children with high probability of inclusion to subgroup 2 (posterior probability of inclusion ≥0.5) compared to those with low probability of inclusion (defined as subgroup 1). Values are median (25^th, 75^th percentile) or n (%). Abbreviations: ALT, alanine aminotransferase; BMI, body mass index; CI, confidence interval; IFN-γ, interferon-γ; IL, interleukin; MCP-1, monocyte chemoattractant protein 1; OR, odds ratio; PAI-1, plasminogen activator inhibitor-1; TNF-α, tumor necrosis factor-α.

FIG. 3.

Hg exposure up-regulates inflammation in human monocyte (THP-1) cells. (A) Cell viability following HgCl₂ exposure at doses of 2.5, 5, 10, 25, and 50 μM for 24 hours and 48 hours. Cytotoxicity was assessed using the Incucyte Live-Cell Analysis System SC3. (B) Transcriptional expression of IL-1β, IL-6, and IL-8 and TNF in monocytes cultured in the presence of HgCl₂ at a dose of 10 μM for 24 hours compared to control. Expression levels were determined in cellular RNA with real-time PCR. (C) Cytokine concentrations in supernatants of monocytes exposed to HgCl₂ (10 μM for 24 hours) compared to control, as determined with the Luminex multiplex platform. Concentrations of IL-1β and IL-6 were below the LOD for this assay (<3 pg/mL). Data are expressed as mean ± SEM. Significant differences were derived by t test with *P < 0.05, **P < 0.01, and ***P < 0.001. Abbreviations: CTL, cytotoxic T lymphocyte; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LOD, limit of detection.