Interactions of Dichlorodiphenyltrichloroethane (DDT) and Dichlorodiphenyldichloroethylene (DDE) With Skeletal Muscle Ryanodine Receptor Type 1

Abstract

Dichlorodiphenyltrichloroethane (DDT) and its metabolite dichlorodiphenyldichloroethylene (DDE) are ubiquitous in the environment and detected in tissues of living organisms. Although DDT owes its insecticidal activity to impeding closure of voltage-gated sodium channels, it mediates toxicity in mammals by acting as an endocrine disruptor (ED). Numerous studies demonstrate DDT/DDE to be EDs, but studies examining muscle-specific effects mediated by nonhormonal receptors in mammals are lacking. Therefore, we investigated whether o,p'-DDT, p,p'-DDT, o,p'-DDE, and p,p'-DDE (DDx, collectively) alter the function of ryanodine receptor type 1 (RyR1), a protein critical for skeletal muscle excitation-contraction coupling and muscle health. DDx (0.01-10 µM) elicited concentration-dependent increases in [3H]ryanodine ([3H]Ry) binding to RyR1 with o,p'-DDE showing highest potency and efficacy. DDx also showed sex differences in [3H]Ry-binding efficacy toward RyR1, where [3H]Ry-binding in female muscle preparations was greater than male counterparts. Measurements of Ca2+ transport across sarcoplasmic reticulum (SR) membrane vesicles further confirmed DDx can selectively engage with RyR1 to cause Ca2+ efflux from SR stores. DDx also disrupts RyR1-signaling in HEK293T cells stably expressing RyR1 (HEK-RyR1). Pretreatment with DDx (0.1-10 µM) for 100 s, 12 h, or 24 h significantly sensitized Ca2+-efflux triggered by RyR agonist caffeine in a concentration-dependent manner. o,p'-DDE (24 h; 1 µM) significantly increased Ca2+-transient amplitude from electrically stimulated mouse myotubes compared with control and displayed abnormal fatigability. In conclusion, our study demonstrates DDx can directly interact and modulate RyR1 conformation, thereby altering SR Ca2+-dynamics and sensitize RyR1-expressing cells to RyR1 activators, which may ultimately contribute to long-term impairments in muscle health.

Keywords: dichlorodiphenyldichloroethylene; dichlorodiphenyltrichloroethane; ryanodine receptors; skeletal muscle.

Figure 1.

The chemical structures of both dichlorodiphenyltrichloroethane (DDT) and dichlorodiphenyldichloroethylene (DDE) congeners: (A), o,p′-DDT; B, p,p′-DDT; (C), o,p′-DDE; (D), p,p′-DDE.

Figure 2.

Concentration-response curves for o,p′-DDT (triangle trace), p,p′-DDT (inverted triangle trace), o,p′-DDE (circle trace), and p,p′-DDE (square trace) with 10 µM bisphenol A (BPA; open circle) as a negative control. The maximum response is significantly different among all 4 congeners (***p < .001). EC₅₀-values are significantly different between the parent compound DDT and its metabolite DDE, but not between congeners within the same group (ie o,p′-DDT vs p,p′-DDT). EC₅₀-values and maximum response were analyzed using a one-way ANOVA with Tukey post hoc test. Two different rabbit JSR membrane preparations were tested, with each membrane run in triplicate (n = 3).

Figure 3.

Both of the dichlorodiphenyltrichloroethane (DDT) and the dichlorodiphenyldichloroethylene (DDE) congeners potentiate Ca²⁺ release from sarcoplasmic reticulum (SR) stores through direct interaction with ryanodine receptor type 1 (RyR1). (A), Representative traces detailing the macroscopic Ca²⁺ efflux assay: rabbit junctional sarcoplasmic reticulum (JSR) vesicles were actively loaded with Ca²⁺ to near maximal capacity and then exposed to either 0.1% DMSO (v/v) vehicle control, 10 µM o,p′-DDT, 10 µM p,p′-DDT, 10 µM o,p′-DDE, or 10 µM p,p′-DDE. Following DMSO or DDx addition, 2 µM ruthenium red, a ryanodine receptor blocker, was added to determine if DDxmediated Ca²⁺ release by direct interaction with RyR1. Lastly, 50 µM cyclopiazonic acid, a sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor, was added to prevent Ca²⁺ reuptake to allow for calibration of Ca²⁺. (B) The initial 60 s of the Ca²⁺ release trace for DMSO vehicle control (square trace), o,p′-DDT (triangle trace), p,p′-DDT (inverted triangle trace), o,p′-DDE (circle trace), or p,p′-DDE (square trace) was assessed with a linear regression to determine Ca²⁺ release rate (nmole Ca²⁺/s/mg JSR). (C), o,p′-DDE (37-fold) and p,p′-DDE (13-fold) significantly increased the rate of Ca²⁺ efflux from Ca²⁺-loaded RyR1 SR vesicles compared with baseline leak rate, whereas both DDT congeners triggered a 6.5-fold difference. Three independent measurements replicated 3 times (n = 3) from different rabbit JSR preparations under identical condition were summarized and plotted (***p < .001; *p < .05, one-way ANOVA with Tukey post hoc test).

Figure 4.

Mouse microsomal preparations from females are more sensitive to [³H]Ry-binding elicited by both dichlorodiphenyltrichloroethane (DDT) and dichlorodiphenyldichloroethylene (DDE) congeners at 10 µM compared with mouse microsomal preparations from males. (A), o,p′-DDT, p,p′-DDT, o,p′-DDE, and p,p′-DDE increased [³H]Ry-binding 3.5- to 6.5-fold higher than 0.1% DMSO (v/v) vehicle control. The same trend on the effects of the DDT and DDE congeners on [³H]Ry-binding was observed when (B) preparations from females (n = 8) were analyzed separately from (C) preparations from males (n = 8). (D) Both DDT and DDE congeners produced a significantly greater effect on [³H]Ry-binding to RyR1 with the preparations from females compared with the preparations from males, which was not attributed to differences in (E) total [³H]Ry basal binding. Experiments were performed in triplicate using 8 membrane preparations per sex from individual mice (n = 8 per sex). Statistical comparison of the effect of DDT and DDE congeners to DMSO vehicle control was performed with a one-way ANOVA with Tukey post hoc test, whereas, comparisons between the effect on the 2 sexes were performed with a Student’s t-test (***p < .001; *p < .05).

Figure 5.

Neither FK506-binding protein (FKBP12) nor ryanodine receptor type 1 (RyR1) protein expression levels are different between preparations from female mice and preparations from male mice. (A), RyR1 microsomal preparations from individual female mice (n = 7) and individual male mice (n = 7) were assessed. (B), Densitometry analysis and a Student’s t-test confirmed no significant differences in protein expression levels of FKBP12, RyR1, or the ratio of RyR1 to FKBP12 between the 2 sexes.

Figure 6.

Acute treatment with either dichlorodiphenyltrichloroethane (DDT) or dichlorodiphenyldichloroethylene (DDE) congeners failed to mediate Ca²⁺ release from ryanodine receptor type 1 (RyR1)-expressing HEK293T cells (HEK-RyR1). (A and B), Fluo-4 fluorescence emission traces showing Ca²⁺-transient responses of HEK-RyR1 cells and wild-type HEK293T cells (HEK-Null) to treatment with various compounds. (A), Immediate addition of 10 µM o,p′-DDT, 10 µM p,p′-DDT, 10 µM o,p′-DDE, 10 µM p,p′-DDE, or 0.1% DMSO (v/v) vehicle control did not facilitate Ca²⁺ release from HEK-RyR1 cells or HEK-Null cells; whereas, addition of 100 µM caffeine, an RyR1 agonist, stimulated Ca²⁺ release from stores in HEK-RyR1 cells but not HEK-Null cells. (B), Treatment of HEK-Null cells with 10 µM thapsigargin, a noncompetitive inhibitor of SERCA, mediated Ca²⁺ release from stores. Experiments were performed in sextuplicate and repeated 4 times (n = 4).

Figure 7.

Dichlorodiphenyltrichloroethane (DDT) and dichlorodiphenyldichloroethylene (DDE) require time to permeate HEK-RyR1 cells to sensitize ryanodine receptor type 1 (RyR1) receptors. (A and B), Fluo-4 fluorescence emission traces showing Ca²⁺-transient responses of HEK-RyR1 cells and HEK-Null cells. (A), Although addition of 100 µM caffeine simultaneously with 10 µM o,p′-DDT, p,p′-DDT, o,p′-DDE, p,p′-DDE, or 0.1% DMSO (v/v) vehicle control does not cause Ca²⁺ release from HEK-Null cells, it stimulates Ca²⁺ release from HEK-RyR1 cells. The degree of RyR1-stimulation by the congeners showed no significant differences compared with DMSO vehicle control as assessed by (C) amplitude and (E) area under the curve (AUC) post-activation. (B), However, addition of 10 µM of either DDT or DDE congener 100 s before addition of 100 µM caffeine sensitized RyR1to the activating effect of 100 µM caffeine, increasing the (D) amplitude and (F) AUC post-stimulation, significantly compared with DMSO vehicle control with caffeine. Experiments were performed in triplicate and repeated 4 times (n = 4). Statistical comparison of the effect of DDT and DDE congeners to DMSO vehicle control was performed with a one-way ANOVA with Dunnett post hoc test (*p < .05; **p < .01; ***p < .001).

Figure 8.

Pretreatment of HEK-RyR1 cells with either dichlorodiphenyltrichloroethane (DDT) or dichlorodiphenyldichloroethylene (DDE) congener for 12-h facilitated a biphasic effect on ryanodine receptor type 1 (RyR1)-sensitization to caffeine response. (A), Ca²⁺-transient response mediated by addition of 100 µM caffeine to HEK-RyR1 cells pretreated with 0.1–10 µM o,p′-DDT (triangle trace), p,p′-DDT (inverted triangle traces), o,p′-DDE (circle traces), or p,p′-DDE (square traces) or 0.1% DMSO (v/v) vehicle control. The (B) amplitude and (C) under the curve (AUC) of the caffeine response were quantified and plotted for each concentration of DDT or DDE. Experiments were performed in triplicate and repeated 4 times (n = 4).

Figure 9.

Pretreatment of HEK-RyR1 cells for 24 h with increasing concentrations of dichlorodiphenyltrichloroethane (DDT) or dichlorodiphenyldichloroethylene (DDE) congeners sensitized HEK-RyR1 cells to caffeine in a dose-dependent manner. (A) Ca²⁺-transient response mediated by addition of 100 µM caffeine to HEK-RyR1 cells pretreated with 0.1–10 µM o,p′-DDT (triangle trace), p,p′-DDT (inverted triangle traces), o,p′-DDE (circle traces), or p,p′-DDE (square traces) or 0.1% DMSO (v/v) vehicle control for 24 h. (B), The area under the curve (AUC) of each trace response were quantified and plotted across all concentrations of DDT and DDE. The analysis of the amplitude of each trace was identical to the AUC (not shown). All DDT and DDE congeners at 5 µM and 10 µM drastically sensitized RyR1 to the activating effect of caffeine and, thus significantly increased amplitude and AUC (***p < .001). Experiments were performed in triplicate and repeated 4 times (n = 4). Statistical comparison of the effect of DDT and DDE congeners to DMSO vehicle control was performed with a one-way ANOVA with Dunnett post hoc test.

Figure 10.

Differentiated myotubes pretreated with 1 µM o,p′-DDE for 24h exhibited heightened sensitivity to electrical stimulation compared with DMSO vehicle control-treated myotubes. Representative traces of the Ca²⁺-transient response mediated by increasing electrical stimulation frequency applied to myotubes pretreated with either 1 µM o,p′-DDE or 0.01% DMSO (v/v) vehicle control. The amplitude of the Ca²⁺ transients of o, p′-DDE pretreated cells (triangle trace) and vehicle pretreated cells (hexagon trace) were quantified and plotted across all electrical stimulation frequency, and a Student’s t-test was performed to determine statistical significance between the 2 groups (*p < .05; **p < .01). Myotubes pretreated with o,p′-DDE displayed greater Ca²⁺-transient amplitudes across all electrical stimulation frequencies compared with vehicle control pretreated cells. Experiments were replicated at least twice using 2 different passages, and all responding control cells (N = 46) and o,p′-DDE pretreated cells (N = 10) were used for analysis. Data shown represent the mean ± SEM.