Comparative Analyses of the 12 Most Abundant PCB Congeners Detected in Human Maternal Serum for Activity at the Thyroid Hormone Receptor and Ryanodine Receptor

Abstract

Polychlorinated biphenyls (PCBs) pose significant risk to the developing human brain; however, mechanisms of PCB developmental neurotoxicity (DNT) remain controversial. Two widely posited mechanisms are tested here using PCBs identified in pregnant women in the MARBLES cohort who are at increased risk for having a child with a neurodevelopmental disorder (NDD). As determined by gas chromatography-triple quadruple mass spectrometry, the mean PCB level in maternal serum was 2.22 ng/mL. The 12 most abundant PCBs were tested singly and as a mixture mimicking the congener profile in maternal serum for activity at the thyroid hormone receptor (THR) and ryanodine receptor (RyR). Neither the mixture nor the individual congeners (2 fM to 2 μM) exhibited agonistic or antagonistic activity in a THR reporter cell line. However, as determined by equilibrium binding of [³H]ryanodine to RyR1-enriched microsomes, the mixture and the individual congeners (50 nM to 50 μM) increased RyR activity by 2.4-19.2-fold. 4-Hydroxy (OH) and 4-sulfate metabolites of PCBs 11 and 52 had no TH activity; but 4-OH PCB 52 had higher potency than the parent congener toward RyR. These data support evidence implicating RyRs as targets in environmentally triggered NDDs and suggest that PCB effects on the THR are not a predominant mechanism driving PCB DNT. These findings provide scientific rationale regarding a point of departure for quantitative risk assessment of PCB DNT, and identify in vitro assays for screening other environmental pollutants for DNT potential.

Figure 1.

The MARBLES mix and its individual components do not activate the THR. Luciferase activity was measured in GH3.TRE-Luc cells treated with either T3 (0.2 nM) or (A) MARBLES mix, (B) PCB 28, 11, 118, (C) PCB 101, 52, 153, (D) PCB 180, 149, 138, or (E) PCB 84, 135, 95. Luciferase activity is expressed as relative light units (RLU) normalized to total protein concentration in the same sample. Data presented as the mean ± SE (n = 5-6 independent experiments). *Significantly different from vehicle (0.1% DMSO) control at p < 0.05, ** p < 0.01, *** p < 0.001 as determined using a one-way ANOVA (p < 0.05) followed by a Holm-Sidak’s multiple comparisons test.

Figure 2.

The MARBLES mix and its individual congeners do not block T3 activation of the THR. Luciferase activity was measured in GH3.TRE-Luc cells treated with T3 (0.2 nM) in the absence or presence of the MARBLES mix (A) or the individual congeners in the mix (B-E). (F) Luciferase activity in cells treated with T3 (0.2 nM) in the absence or presence of the THR blocker, NH-3 (100 pM). Luciferase activity is expressed as relative light units (RLU) normalized to total protein concentration. Data presented as the mean ± SE (n = 6 independent experiments). *Significantly different from vehicle (0.1% DMSO) at p < 0.05, ** p < 0.01, *** p < 0.001, †Significantly different from T3 at p < 0.01 as determined using a one-way ANOVA (p < 0.05) and post hoc Holm-Sidak’s multiple comparisons test.

Figure 3.

The MARBLES mix and its individual components do not antagonize T4-induced THR signaling. Luciferase activity was measured in GH3.TRE-Luc cells treated with T4 (2 nM) in the absence or presence of the MARBLES mix (A) or the individual congeners in the mix (B-E). (F) Luciferase activity in cells treated with T4 (2 nM) in the absence or presence of the THR blocker, NH-3 (100 nM). Luciferase activity is expressed as relative light units (RLU) normalized to total protein concentration of each sample. Data presented as the mean ± SE (n = 5-6 independent experiments). *Significantly different from vehicle (0.1% DMSO) at p < 0.05, ** p < 0.01, *** p < 0.001, †Significantly different from T4 at p < 0.0001 as determined using a one-way ANOVA (p < 0.05) and post hoc Holm-Sidak’s multiple comparisons test.

Figure 4.

PCB 11 and 52 metabolites exhibit no agonistic or antagonistic activity at the THR. (A) To test for THR agonism, luciferase activity was measured in GH3.TRE-Luc cells treated with either T3 (0.2 nM) or metabolites of PCB 11 or 52. (B-C) To test for THR antagonism, luciferase activity was measured in cells treated with 0.2 nM T3 (B) or 2 nM T4 (C) in the absence or presence of PCB 11 or 52 metabolites. Luciferase activity is expressed as relative light units (RLU) normalized to total protein concentration. Data presented as the mean ± SE (n = 4-6 independent experiments). *Significantly different from vehicle (0.1% DMSO) at p < 0.05, ** p < 0.01, *** p < 0.001 as determined using one-way ANOVA (p < 0.05) with post hoc Holm-Sidak’s multiple comparisons test.

Figure 5.. The MARBLES mix and its 12 individual congeners allosterically enhance the binding of [³H]Ry to RyR1 in its high affinity open conformational state.

Specific [³H]Ry binding in the presence of the MARBLES mix (A), individual PCB congeners (B-E) or vehicle (DMSO, 1%). DMSO concentrations in PCB samples varied from 1% to 0.2%. Data was plotted as fold increase from respective baseline binding. Baseline binding in the presence of DMSO (1%) averaged 0.16±0.06 pmol/mg protein across experimental trials. Each data point represents the mean ± SE of triplicate determinations repeated 2-5 times under identical experiment conditions. Concentration-effect data were fitted using nonlinear regression Sigmoidal Boltzmann or Logistic with Origin 9.1, and the latter was used to obtain EC₅₀ values and their maximal activation levels (a measure of efficacy), which is summarized in Table 3.

Figure 6:

The parent congener and 4-OH and sulfate metabolites of PCB 11 (A) and PCB 52 (B) were tested using [³H]Ry binding analysis as described in Figure 5. Data represent 12 replicates performed on at least 4 independent measurements and are plotted as fold-increase from DMSO control. Baseline binding average is 0.14±0.01pmol/mg protein).