Exposure to Trichloroethylene Metabolite S-(1,2-Dichlorovinyl)-L-cysteine Causes Compensatory Changes to Macronutrient Utilization and Energy Metabolism in Placental HTR-8/SVneo Cells

Abstract

Trichloroethylene (TCE) is a widespread environmental contaminant following decades of use as an industrial solvent, improper disposal, and remediation challenges. Consequently, TCE exposure continues to constitute a risk to human health. Despite epidemiological evidence associating exposure with adverse birth outcomes, the effects of TCE and its metabolite S-(1, 2-dichlorovinyl)-L-cysteine (DCVC) on the placenta remain undetermined. Flexible and efficient macronutrient and energy metabolism pathway utilization is essential for placental cell physiological adaptability. Because DCVC is known to compromise cellular energy status and disrupt energy metabolism in renal proximal tubular cells, this study investigated the effects of DCVC on cellular energy status and energy metabolism pathways in placental cells. Human extravillous trophoblast cells, HTR-8/SVneo, were exposed to 5-20 μM DCVC for 6 or 12 h. After establishing concentration and exposure duration thresholds for DCVC-induced cytotoxicity, targeted metabolomics was used to evaluate overall energy status and metabolite concentrations from energy metabolism pathways. The data revealed glucose metabolism perturbations including a time-dependent accumulation of glucose-6-phosphate+frutose-6-phosphate (G6P+F6P) as well as independent shunting of glucose intermediates that diminished with time, with modest energy status decline but in the absence of significant changes in ATP concentrations. Furthermore, metabolic profiling suggested that DCVC stimulated compensatory utilization of glycerol, lipid, and amino acid metabolism to provide intermediate substrates entering downstream in the glycolytic pathway or the tricarboxylic acid cycle. Lastly, amino acid deprivation increased susceptibility to DCVC-induced cytotoxicity. Taken together, these results suggest that DCVC caused metabolic perturbations necessitating adaptations in macronutrient and energy metabolism pathway utilization to maintain adequate ATP levels.

Figure 1

DCVC cytotoxicity. HTR-8/SVneo cells were treated for 12, 24, or 48 h with medium alone (control), or with 5, 10, or 20 μM DCVC. The MultiTox-Glo Multiplex Cytotoxicity Kit (Promega) was used to measure the relative number of live and dead cells within a single well as described in the Experimental Procedures. Graphical representation shows live-to-dead cell ratios as percent control within each time point. Bars represent means ± SEM. Data were analyzed by two-way ANOVA (interaction between time and treatment, P < 0.0001) with post hoc Tukey multiple comparisons. Pound sign indicates significant difference compared to same treatment at all earlier time points: ^#P < 0.0001. At symbol indicates significant difference compared to same treatment at 12 h time point: ^@P < 0.03. Asterisk indicates significant difference compared to medium alone (control) within same time point: *P = 0.0008. Plus sign indicates significant difference compared to control and 5 μM DCVC within same time point: ⁺P < 0.02. N = 3 independent experiments for each time point, with three replicates per treatment in each experiment. Camptothecin (4 μM) was included as a positive control and decreased the live-to-dead cell ratio by 55.6% ± 2.17% at 12 h, 80.68% ± 0.531% at 24 h, and 32.89% ± 0.039%% at 48 h.

Figure 2

DCVC-induced changes in key cellular energy status indictors. Targeted metabolomics analysis was used to measure concentrations of energy status metabolites in HTR-8/SVneo cells treated with medium alone (control) or 20 μM DCVC for 6 or 12 h. (A) Graphical representations of concentrations of: (i) adenylate and guanylate nucleotides, (ii) phosphate donor and product phosphocreatine and creatine, and (iii) electron transporters NAD+ and NADH. Boxes represent first quartile, median, and third quartile; whiskers represent minimum and maximum concentrations. (B) Graphical representations of energy metabolite ratios derived from metabolite concentrations: (i) ATP:AMP, (ii) ATP:ADP, (iii) phosphocreatine:creatine, and (iv) NADH:NAD+. Bars represent ratio means ± SEM. All data were analyzed by two-way ANOVA (interaction between time and treatment varied by metabolite, P < 0.05) with post hoc Tukey multiple comparisons. Asterisks indicate significant differences compared to medium alone (control): *P < 0.0419, **P < 0.0097, ***P < 0.001. Pound signs indicates significant differences compared to same treatment at all earlier time points: #P = 0.0116, ^##P = 0.0026, ^###P < 0.001. N = 5 independent experiments for each time point. (C) AMPK signaling pathway was evaluated with western blotting analysis and normalized to total protein. (i) Graphical representation of p-AMPKα:p-AMPKα ratio and (ii) representative western blotting images. Bars represent means ± SEM. Data were analyzed with student t tests. N = 3 independent experiment, with three replicates per treatment in each experiment.

Figure 3

Effects of DCVC on energy metabolism pathways. HTR-8/SVneo cells were treated with medium alone (control) or 20 μM DCVC for 6 or 12 h. Targeted metabolomics analysis was used to measure a panel of intracellular metabolites unique to specific energy metabolism pathways. (A) Overview of DCVC-induced changes in integrated energy metabolism pathways. Blue arrows indicate pathway directionality. Metabolite names in red indicate altered concentrations between treatment groups within same time point (P < 0.05). Purple and pink arrows indicate direction of change in concentrations within 6 or 12 h time points, respectively. Green star symbols indicate altered concentrations between time points within same treatment group (P < 0.05). All other symbols are indicated in figure legend. (B) Graphical representations of selected metabolite concentrations grouped by energy metabolic pathway. Background color indicates corresponding pathway on integrated overview in panel A. Pathways represented include: (i) glucose metabolism, (ii) pentose phosphate pathway, (iii) purine pathways, (vi) hexosamine biosynthesis pathway, (v) glycolysis, (vi) TCA cycle pathway, (vii) glycerol metabolism pathway, (viii) β-oxidation pathway, and (ix) amino acid metabolism pathways. Within each graph, boxes represent first quartile, median, and third quartile; whiskers represent minimum and maximum. All data were log2 transformed prior to statistical analysis to achieve normal Gaussian distribution. Data were analyzed by two-way ANOVA (interaction between time and treatment varied depending on metabolite, P < 0.05) with post hoc Tukey multiple comparisons. Asterisks indicate significant differences compared to medium alone (control): *P < 0.05, **P < 0.01, ***P < 0.001. Pound signs indicate significant differences compared to same treatment at all earlier time points: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. N = 5 independent experiments for each time point. (C) Graphical representation of phosphofructokinase 1 activity after 12 h DCVC treatment. PFK1 activity was measured with a commercially available enzyme activity assay kit (Sigma-Aldrich). Bars represent means ± SEM. Data were analyzed with student t test. N = 3 independent experiments, with three replicates per treatment in each experiment.

Figure 3

Effects of DCVC on energy metabolism pathways. HTR-8/SVneo cells were treated with medium alone (control) or 20 μM DCVC for 6 or 12 h. Targeted metabolomics analysis was used to measure a panel of intracellular metabolites unique to specific energy metabolism pathways. (A) Overview of DCVC-induced changes in integrated energy metabolism pathways. Blue arrows indicate pathway directionality. Metabolite names in red indicate altered concentrations between treatment groups within same time point (P < 0.05). Purple and pink arrows indicate direction of change in concentrations within 6 or 12 h time points, respectively. Green star symbols indicate altered concentrations between time points within same treatment group (P < 0.05). All other symbols are indicated in figure legend. (B) Graphical representations of selected metabolite concentrations grouped by energy metabolic pathway. Background color indicates corresponding pathway on integrated overview in panel A. Pathways represented include: (i) glucose metabolism, (ii) pentose phosphate pathway, (iii) purine pathways, (vi) hexosamine biosynthesis pathway, (v) glycolysis, (vi) TCA cycle pathway, (vii) glycerol metabolism pathway, (viii) β-oxidation pathway, and (ix) amino acid metabolism pathways. Within each graph, boxes represent first quartile, median, and third quartile; whiskers represent minimum and maximum. All data were log2 transformed prior to statistical analysis to achieve normal Gaussian distribution. Data were analyzed by two-way ANOVA (interaction between time and treatment varied depending on metabolite, P < 0.05) with post hoc Tukey multiple comparisons. Asterisks indicate significant differences compared to medium alone (control): *P < 0.05, **P < 0.01, ***P < 0.001. Pound signs indicate significant differences compared to same treatment at all earlier time points: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. N = 5 independent experiments for each time point. (C) Graphical representation of phosphofructokinase 1 activity after 12 h DCVC treatment. PFK1 activity was measured with a commercially available enzyme activity assay kit (Sigma-Aldrich). Bars represent means ± SEM. Data were analyzed with student t test. N = 3 independent experiments, with three replicates per treatment in each experiment.

Figure 4

DCVC-induced changes in amino acid transporter levels. Energy-relevant amino acid transporter levels in HTR-8/SVneo cells treated with medium alone (control) or 20 μM DCVC for 12 h were evaluated with western blotting analysis and normalized to total protein. (A) Small neutral amino acid transporter ASCT2/SLC1A5. (B) Large amino acid transporter heavy subunit 4F2hc/SLC3A2. (C) Large neutral amino acid transporter LAT1/SLC7A5. (D) Representative western blot images. Bars represent means ± SEM as percent control. Data were analyzed with student t tests. Asterisks indicate significant difference compared to medium alone (control): * P = 0.0474. N = 3 independent experiments, with three replicates per treatment in each experiment.

Figure 5

Effects of amino acid deprivation on DCVC-induced cytotoxicity. HTR-8/SVneo cells were cultured in DMEM containing the following proportions of amino acid-free medium: 0%, 20%, 40%, 60%, 80%, and 100%, mixed with respective proportions of amino acid-containing DMEM medium. Within each culture condition, cells were also treated in triplicate with medium alone (control) or 20 μM DCVC for 12 h. The MultiTox-Glo Multiplex Cytotoxicity Kit (Promega) was used to measure live and dead cells, as previously described in the Experimental Procedures. Graphical representation shows live-to-dead cell ratios as a percentage of nontreated controls within each respective cell culture condition group. Bars represent means ± SEM. Data were analyzed by two-way ANOVA (interaction between cell culture conditions and DCVC treatment, P < 0.0001) with post hoc Tukey multiple comparisons. Asterisks indicate significant differences compared to medium alone (control) within respective cell culture conditions groups: *P < 0.0002. Dollar symbol indicates significant difference compared to same DCVC treatment cultured with amino acid-free medium: ^$P = 0.0137. N = 3 independent experiments, with three replicates per treatment and cell culture condition groups in each experiment.

Figure 6

Energy pathway metabolite ratios. Graphical representations of energy metabolite ratio calculated from metabolite concentrations representing upstream glycolysis, pentose phosphate pathway, and glycerol metabolism pathway. (A) Glucose:G6P+F6P ratio, (B) G6P+F6P:R5P+X5P ratio, (C) G6P+F6P:R5P+X5P ratio, (D) G6P+F6P:NAcG1P ratio, (E) F1BP:Ga3P+DHAP ratio, (F) Ga3P+DHAP:2G+3PG ratio, (G) Ga3P+DHAP:GL3P ratio. Metabolite names in dark red indicate metabolites with noteworthy DCVC-induced fluctuations. Boxes within each graph represent first quartile, median, and third quartile; whiskers represent minimum and maximum. All data were log2 transformed prior to statistical analysis to achieve normal Gaussian distribution. Data were analyzed by two-way ANOVA (interaction between time and treatment varied depending on metabolite, P < 0.05) with post hoc Tukey multiple comparisons. Asterisks indicate significant differences compared to medium alone (control): *P < 0.05, **P < 0.01, ***P < 0.001. Pound signs indicate significant differences compared to same treatment at all earlier time points: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

Figure 7

Proposed DCVC-induced energy metabolism alterations HTR-8/SVneo cells. (A) Overview of normal cellular energy metabolism pathways in first-trimester placental cells. First-trimester placental cells survive in a low-oxygen environment. As a result, these cells prefer glycolysis fueled by glucose as their primary source of energy over oxidative phosphorylation. Despite this preference, the cells are capable of using other macronutrient metabolism pathways to fuel oxidative phosphorylation for additional ATP synthesis. (B) Summary of proposed DCVC-induced energy metabolism alterations in HTR-8/SVneo cells. 1: Following glucose phosphorylation, G6P+F6P accumulated in a time-dependent manner. Conversely, PPP and HBP shunting of G6P+F6P was elevated at 6 h and diminished with time, suggesting two independent processes. 2: At 6 and 12 h, alternative bioenergentic fuel sources and pathways including amino acid, lipid, and glycerol metabolism provided intermediates that enter glycolysis downstream of the G6P+F6P accumulation or enter the TCA cycle as acetyl CoA. Additionally, Ga3P and DHAP concentrations were elevated at both time points, suggesting another possible glycolytic perturbation. 3: Acetyl-CoA concentrations were increased at both time points, but TCA cycle metabolites were largely unchanged, indicating that DCVC likely does not directly affect the TCA cycle. 4: Although ATP levels are sustained, adenylate nucleotide ratios shifted down, and ADP and AMP concentrations increased.