Cell death mechanisms in GT1-7 GnRH cells exposed to polychlorinated biphenyls PCB74, PCB118, and PCB153

Abstract

Exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) causes functional deficits in neuroendocrine systems. We used an immortalized hypothalamic GT1-7 cell line, which synthesizes the neuroendocrine peptide gonadotropin-releasing hormone (GnRH), to examine the neurotoxic and endocrine disrupting effects of PCBs and their mechanisms of action. Cells were treated for 1, 4, 8, or 24 h with a range of doses of a representative PCB from each of three classes: coplanar (2,4,4',5-tetrachlorobiphenyl: PCB74), dioxin-like coplanar (2',3,4,4',5' pentachlorobiphenyl: PCB118), non-coplanar (2,2',4,4',5,5'-hexachlorobiphenyl: PCB153), or their combination. GnRH peptide concentrations, cell viability, apoptotic and necrotic cell death, and caspase activation were quantified. In general, GnRH peptide levels were suppressed by high doses and longer durations of PCBs, and elevated at low doses and shorter timepoints. The suppression of GnRH peptide levels was partially reversed in cultures co-treated with the estrogen receptor antagonist ICI 182,780. All PCBs reduced viability and increased both apoptotic and necrotic cell death. Although the effects for the three classes of PCBs were often similar, subtle differences in responses, together with evidence that the combination of PCBs acted slightly different from individual PCBs, suggest that the three tested PCB compounds may act via slightly different or more than one mechanism. These results provide evidence that PCB congeners have endocrine disrupting and/or neurotoxic effects on the hypothalamic GnRH cell line, a finding that has implications for environmental endocrine disruption in animals.

Figure 1

The effects of (A) PCB74, (B) PCB118, (C) PCB153, or (D) PCB Mix on GT1-7 GnRH peptide levels. The cells, seeded on 96-well plates, were treated for 1, 4, 8, and 24 hrs with 0.1% DMSO alone (vehicle) or with 0.1, 1, 10, or 100 μM PCB. By 4 hr post-treatment, a stimulation of peptide was observed in PCB-treated cells in a dose-dependent manner that varied by compound. At late time points (8 hr, 24 hr) PCBs suppressed GnRH peptide. Results are expressed as average GnRH peptide levels ± S.E.M for each experimental average of pooled triplicate cultures (N = 4) per treatment. *p < 0.05, **p < 0.005 vs. vehicle (DMSO).

Figure 2. Estrogen receptor antagonism modulates PCB-suppression of GnRH peptide release

Cells were treated for 24 hrs with 0.1% DMSO alone (vehicle), 1000 nM ICI alone, 1 μM PCBs, or 1 μM PCBs coadministered with ICI. Coadministration of PCB with ICI partially reversed PCB-induced suppression of GnRH peptide. Results are expressed as average GnRH peptide levels ± S.E.M for each experimental average of pooled triplicate cultures (N = 4) per treatment. *p < 0.05, **p < 0.005 vs. vehicle (DMSO).

Figure 3

The effects of (A) PCB74, (B) PCB118, (C) PCB153, and (D) PCB Mix on GT1-7 cell viability. Fluorescence measured at 560_Ex/590_Em is proportional to the number of metabolically active cells. Samples at each timepoint were normalized to vehicle control fluorescence at 1 hr timepoint, and are expressed as percentage control. Individual congeners had inhibitory effects upon cell metabolism/viability vs. vehicle at 1 hr, whereas their combination only differed from control at the lowest dose at 4 hr. Each value, expressed as percentage control, is the mean ± S.E.M of four experiments run in triplicate. After 24 hrs treatment, all PCBs decreased viability. *p < 0.05, **p < 0.005 vs. vehicle (DMSO) at 1 hr baseline.

Figure 4

Fluorescence assays of markers of cell death are shown. (A) Trypan Blue staining of GT1-7 cells is shown for a representative PCB118 treated culture. Normal cells appear unstained and necrotic cells (arrow) appear blue. (B) DAPI staining of GT1-7 nuclei is shown in a representative PCB74 treated culture. The arrow shows a condensed apoptotic nucleus, and the arrowhead shows a mitotic (non-apoptotic) nucleus. Scale bar = 10 μm (panels A and B). (C and D) Cleaved caspase-9 immunofluorescence is shown for the 4 hour timepoint for a representative control (C) or PCB Mix (D) culture. Results for individual PCBs and the PCB Mix were similar, and all PCBs activated cleaved caspase-9 immunofluorescence. (E and F) Cleaved caspase-8 immunofluorescence is shown for the 4 hour timepoint for a representative control (C) or PCB Mix (D) culture. Results for individual PCBs were indistinguishable from the PCB Mix (not shown). Caspase-8 immunofluorescence was not detectable in any of the PCB- or vehicle-treated cultures. Scale bar = 10 μm (panels C – F).

Figure 5

The effects of (A) PCB74, (B) PCB118, (C) PCB153, and (D) PCB Mix treatment upon GT1-7 trypan blue staining (necrotic cell death). Cells were incubated in absence (DMSO) or presence of PCB concentrations (0.1, 1, 10, or 100 μM) for different durations (1, 4, 8, 24 hr). Results are expressed as a percentage of blue stained cells characteristic of necrotic cells ± S.E.M for 4 triplicates of each experimental condition. We observed a slight but significant increase in necrotic cell death after 8 h treatment with the highest dose PCB118 or PCB153. After 24 h treatment, the highest dose PCB74 and two highest doses of PCB118 and PCB153 caused an extensive increase in necrotic cell death.

Figure 6

The effects of (A) PCB74, (B) PCB118, (C) PCB153, and (D) PCB Mix on GT1-7 apoptotic cell death. Cells were incubated in absence (DMSO) or presence of PCB concentrations (0.1, 1, 10, or 100 μM) for different durations (1, 4, 8, 24 hr). Results are expressed as a percentage pyknotic DAPI stained cells characteristic of apoptotic cells ± S.E.M for 4 triplicates of each experimental condition. We observed a significant increase in apoptotic cell death as early as 1 hr treatment with lower doses (0.1 or 1 μM) PCB74, PCB118, or PCB Mix. After 24 h treatment, all PCBs caused a significant increase in apoptotic cell death.

Figure 7

The effects of (A) PCB74, (B) PCB118, (C) PCB153, or (D) PCB Mix on caspase-3/7 activation. Cells were incubated in absence (DMSO) or presence of PCB concentrations (0.1, 1, 10, or 100 μM) for four hours. Results are expressed as fold increase of caspase-3/7 activation compared to control cells ± S.E.M for 4 triplicates of each experimental condition. Treatment with PCB74, PCB118 or the PCB Mix resulted in activation of caspase-3/7, while PCB153 did not significantly affect caspase-3/7 activity. *p < 0.05, **p < 0.005 vs. vehicle (DMSO) at 4 hr.