Triclocarban enhances testosterone action: a new type of endocrine disruptor?

Abstract

Many xenobiotics have been associated with endocrine effects in a wide range of biological systems. These associations are usually between small nonsteroid molecules and steroid receptor signaling systems. In this report, triclocarban (TCC; 3,4,4'-trichlorocarbanilide), a common ingredient in personal care products that is used as an antimicrobial agent was evaluated and found to represent a new category of endocrine-disrupting substance. A cell-based androgen receptor-mediated bioassay was used to demonstrate that TCC and other urea compounds with a similar structure, which have little or no endocrine activity when tested alone, act to enhance testosterone (T)-induced androgen receptor-mediated transcriptional activity in vitro. This amplification effect of TCC was also apparent in vivo when 0.25% TCC was added to the diet of castrated male rats that were supported by exogenous testosterone treatment for 10 d. All male sex accessory organs increased significantly in size after the T+TCC treatment, compared with T or TCC treatments alone. The data presented here suggest that the bioactivity of endogenous hormones may be amplified by exposure to commercial personal care products containing sufficient levels of TCC.

Figure 1

Structure of TCC.

Figure 2

The effect of TCC on cell proliferation. 2933Y cells were treated for 16 h with TCC (1.0 μm) alone or in combination with T (0.125 nm). The cytotoxicity or cell proliferation was evaluated by the MTT assay. The absorbance was measured at 570 nm with a reference wavelength of 650 nm using an EMax spectrophotometer (Molecular Devices, Sunnyvale, CA). Absorbance (OD) at 570 nm is expressed as mean ± sd (n = 6). No significant difference in cell proliferation was observed in cells with TCC alone or in combination with T when compared with the vehicle control.

Figure 3

The effect of TCC on AR-mediated transcriptional activity induced by T. 2933Y cells were treated for 16 h with and without TCC (1.0 μm) and in combination with T (0.125 nm) and/or flutamide (10 μm). Cell lysates were assessed for luciferase activity, which is expressed as mean ± sd (n = 4) of relative light units (RLU). a, Significantly different from vehicle control; b, significantly different from vehicle control and T treatment.

Figure 4

A, The time course effect of TCC on AR-mediated transcriptional activity induced by T. 2933Y cells were treated with T (0.125 nm) alone (•) or with a combination of TCC (1.0 μm) (○) for different time periods. B, The dose-response effect of TCC on AR-mediated transcriptional activity induced by T. 2933Y cells were treated with T (0–1.0 nm) alone (•) or with a combination of TCC (1.0 μm) (○) for 16 h. C, The dose-response effect of TCC on AR-mediated transcriptional activity induced by T. 2933Y cells were treated with T (0.125 nm) alone or with a combination of various concentrations of TCC for 16 h. In each experiment, cell lysates were measured for luciferase activity, which is expressed as mean ± sd (n = 3) of relative light units (RLU). *, Significantly different from T treatment.

Figure 5

The augmentation of TCC analogs on AR-mediated transcriptional activity. Closed bar, T at 0.125 nm alone; open bar, vehicle control; checkered bar, TCC analogs at 1.0 μm alone; hatched bar, TCC analogs at 1.0 μm in the presence of 0.125 nm of T. 1, Carbanilide; 2, 4,4′-dichlorocarbanilide; 3, TCC; 4, 3,3′,4,4′-tetrachlorocarbanilide; 5, 4′-methoxy-3,4-dichlorocarbanilide.

Figure 6

Competitive binding of TCC in AR FP assay. Rat AR ligand binding domain/fluormone complex was incubated with TCC at various concentrations. A, Maximum FP in the absence of any competitor. B and C, FP values in the presence of DHT, a strong AR competitor (B: 10 nm and C: 100 nm). D-I, FP values in the presence of increasing concentrations of TCC (D: 2 nm; E: 20 nm; F: 200 nm; G: 2 μm; H: 20 μm; I: 200 μm). Data presented as mean ± sd of triplicates.

Figure 7

Effect of TCC on the amount of immunoreactive AR protein. MDA-kb2 or 2933Y cells were treated with vehicle, T (1.0 nm), TCC (1.0 μm), or a combination of T+TCC for 48 h. Whole-cell lysates were probed by Western blot analysis with antibody against amino acids 299–315 of human AR. Each lane contained either 60 μg (for MDA-kb2) or 15 μg (for 2933Y) of protein. Veh, Vehicle control.

Figure 8

The effect of TCC on cAMP/PKA-mediated transcriptional activity induced by hCG. JK293 cells were treated for 16 h with and without TCC (1.0 μm) and in combination with hCG (3.2 ng/ml), T (0.125 nm), and/or flutamide (10 μm). Cell lysates were measured for luciferase activity, which is expressed as mean ± sd (n = 4) of relative light units (RLU). Neither T nor flutamide induced any effect on cAMP/PKA-mediated transcriptional activity. No significant differences in luciferase activities were observed in TCC and hCG combinations when compared with hCG treatment alone.