In vitro biologic activities of the antimicrobials triclocarban, its analogs, and triclosan in bioassay screens: receptor-based bioassay screens

Abstract

Background: Concerns have been raised about the biological and toxicologic effects of the antimicrobials triclocarban (TCC) and triclosan (TCS) in personal care products. Few studies have evaluated their biological activities in mammalian cells to assess their potential for adverse effects.

Objectives: In this study, we assessed the activity of TCC, its analogs, and TCS in in vitro nuclear-receptor-responsive and calcium signaling bioassays.

Materials and methods: We determined the biological activities of the compounds in in vitro, cell-based, and nuclear-receptor-responsive bioassays for receptors for aryl hydrocarbon (AhR), estrogen (ER), androgen (AR), and ryanodine (RyR1).

Results: Some carbanilide compounds, including TCC (1-10 muM), enhanced estradiol (E(2))-dependent or testosterone-dependent activation of ER- and AR-responsive gene expression up to 2.5-fold but exhibited little or no agonistic activity alone. Some carbanilides and TCS exhibited weak agonistic and/or antagonistic activity in the AhR-responsive bioassay. TCS exhibited antagonistic activity in both ER- and AR-responsive bioassays. TCS (0.1-10 muM) significantly enhanced the binding of [(3)H]ryanodine to RyR1 and caused elevation of resting cytosolic [Ca(2+)] in primary skeletal myotubes, but carbanilides had no effect.

Conclusions: Carbanilides, including TCC, enhanced hormone-dependent induction of ER- and AR-dependent gene expression but had little agonist activity, suggesting a new mechanism of action of endocrine-disrupting compounds. TCS, structurally similar to noncoplanar ortho-substituted poly-chlorinated biphenyls, exhibited weak AhR activity but interacted with RyR1 and stimulated Ca(2+) mobilization. These observations have potential implications for human and animal health. Further investigations are needed into the biological and toxicologic effects of TCC, its analogs, and TCS.

Keywords: androgen receptor; antimicrobial; aryl hydrocarbon receptor; bioactivity; carbanilide analog; estrogen receptor; ryanodine receptor; sensitization; signal amplification; triclocarban; triclosan.

Figure 1

Chemical structures and use of TCC, its analogs, and TCS.

Figure 2

Results of ER- and AR-mediated bioassays showing the effects of 1 μM TCC on gene expression of ER (A) and AR (B) induced by E₂ in BG1-ERE cells and testosterone (T) in T47D-ARE cells, respectively. Luciferase activity (mean ± SD) is expressed relative to that maximally induced by E₂ and T in (A) and (B), respectively. *Significantly greater than E₂ or T positive control groups (p < 0.05).

Figure 3

Effects of carbanilide compounds at 1 (10⁻⁶ M) and 10 μM (10⁻⁵ M) on ER- and AR-mediated activity in the absence (A and C) and presence (B and D) of E₂ (1 nM; A and B) or testosterone (T; 10 μM; C and D) in ER-responsive (BG1-ERE cells) or AR-responsive (T47D-ARE cells) bioassay. *Significantly greater than the solvent control (A and C) or E₂ or T positive controls (B and D) at p < 0.05 for agonist/amplification evaluation. ^#Cell toxicity at 10-μM concentration.

Figure 4

Effects of 1-μM carbanilide compounds on the AR-mediated activity in the absence (A) and the presence (B) of testosterone (T; 0.1 nM) in the HEK-2933Y AR-responsive cell system. The activity by carbanilide compounds I–V was reported by Chen et al. (2008). *Significantly greater than the solvent control (A) or T positive control (B) at p < 0.05 for agonist/amplification evaluation.

Figure 5

The effects of TCC (III) tested at different concentrations on ER (A) or AR (B) gene expression in the presence of steroid hormone E₂ (1 nM) or testosterone (T; 10 μM) at a constant concentration in the ER-responsive (BG1-ERE; A) or AR-responsive (T47D-ARE; B) bioassay. *Significantly greater than the E₂ or T positive control at p < 0.05.

Figure 6

Activity of TCS in the ER-mediated bioassay. *Significantly different from the control.

Figure 7

[³H]Ry binding with or without 1.2 μM TCS in skeletal muscle sarcoplasmic reticulum vesicles. *Significantly greater than the control at p < 0.05.

Figure 8

Effect of TCS on cytosolic Ca²⁺ concentration. (A) Cytosolic Ca²⁺ concentration in resting myotubes increased in a dose-dependent manner after TCS treatment; each trace is an average of n ≥ 5 cells in separate cell cultures in Ca²⁺-replete (1.8 mM) buffer. (B) TCS 1 μM triggered an increase in the cytosolic Ca²⁺ concentration even in nominally Ca²⁺-free (~ 7 μM) extracellular buffer.

Figure 9

Three-dimensional projection of TCS and PCB-95 generated by ChemIDplus (National Library of Medicine 2008).