Antimicrobial agent triclosan is a proton ionophore uncoupler of mitochondria in living rat and human mast cells and in primary human keratinocytes

Abstract

Triclosan (TCS) is an antimicrobial used widely in hospitals and personal care products, at ~10 mM. Human skin efficiently absorbs TCS. Mast cells are ubiquitous key players both in physiological processes and in disease, including asthma, cancer, and autism. We previously showed that non-cytotoxic levels of TCS inhibit degranulation, the release of histamine and other mediators, from rat mast cells (RBL-2H3), and in this study we replicate this finding in human (HMC-1.2) mast cells. Our investigation into the molecular mechanisms underlying this effect led to the discovery that TCS disrupts ATP production in RBL-2H3 cells in glucose-free, galactose-containing media (95% CI EC50 = 7.5-9.7 μM), without causing cytotoxicity. Using these same glucose-free conditions, 15 μM TCS dampens RBL-2H3 degranulation by 40%. The same ATP disruption was found with human HMC-1.2 cells (EC50 4.2-13.7 μM), NIH-3T3 mouse fibroblasts (EC50 4.8-7.4 μM), and primary human keratinocytes (EC50 3.0-4.1 μM) all with no cytotoxicity. TCS increases oxygen consumption rate in RBL-2H3 cells. Known mitochondrial uncouplers (e.g., CCCP) previously were found to inhibit mast cell function. TCS-methyl, which has a methyl group in place of TCS’s ionizable proton, affects neither degranulation nor ATP production at non-cytotoxic doses. Thus, triclosan’s effects on mast cell function are due to its proton ionophore structure. Also, 5 μM TCS inhibits thapsigargin-stimulated degranulation of RBL-2H3 cells: further evidence that TCS disrupts mast cell signaling. Our data indicate that TCS is a mitochondrial uncoupler, and TCS may affect numerous cell types and functions via this mechanism.

Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening

“…Hits belonged to three main pharmacological classes (Figure 3F), anti-psychotics, antidepressants, and antihypertensives, all of which are consistently represented in small molecule libraries (Attene-Ramos et al, 2015; Gohil et al, 2010; Stavrovskaya et al, 2004). The majority of hits have been shown to uncouple mt-Δψ (e.g., carvedilol, triclosan, sertraline, and hexachlorophene) (Cammer and Moore, 1972; Oliveira et al, 2000; Weatherly et al, 2016), to induce mitochondrial swelling (e.g., meclomen and benzbromarone) (Tatematsu et al, 2016), and to impair ETC and OXPHOS (e.g., miconazol, tamoxifen, trifluoperazine, and mefloquine) (Cheah and Waring, 1983; Dickinson, 1977; Gohil et al, 2010; Tuquet et al, 2000). The remaining compounds included a monoamine transporter inhibitor (indatraline), two plasma membrane voltage-gated Ca 2+ channel blockers (amlodipine and nicardipine), and one 5-HT receptor agonist (tegaserod).…”

Triclosan exposure, transformation, and human health effects

“…Multiplying this result by the factors noted above, (1 mg cell/0.2 mg protein) and (cell/3.5×10 −6 mg), yields approximately 2.8 nmol of TCS is absorbed per mg of mammalian protein within the cells. Thus, approximately 2.8 nmol TCS exposure/mg cellular protein induced mitotoxicity in rat basophilic leukemia (RBL) mast cells (Weatherly et al 2016). Specifically using primary human keratinocytes, a significant decrease was noted in mitochondrial ATP production due to an exposure of 3 μM TCS (Weatherly et al 2016), which corresponds to approximately 0.8 nmol/mg protein .…”

“…Thus, approximately 2.8 nmol TCS exposure/mg cellular protein induced mitotoxicity in rat basophilic leukemia (RBL) mast cells (Weatherly et al 2016). Specifically using primary human keratinocytes, a significant decrease was noted in mitochondrial ATP production due to an exposure of 3 μM TCS (Weatherly et al 2016), which corresponds to approximately 0.8 nmol/mg protein . These levels may be overestimates of the amount of TCS that permeates cells because absorption may be less than 10% of the total amount since 200 μl is a large volume compared to the volume of 100,000 cells.…”

“…This topic was recently reviewed by Ruszkiewicz et al (2017). Briefly, TCS was found to affect mitochondrial function (Weatherly et al 2016; Shim et al 2016; Newton et al 2005; Ajao et al 2015), disrupt proper calcium signaling (Cherednichenko et al 2012), alter zinc homeostasis (Tamura et al 2012), and affect immunological parameters (Anderson et al 2013; Yueh et al 2014; Udoji et al 2010; Weatherly et al 2013). …”

Effect of contaminants of emerging concern on liver mitochondrial function in Chinook salmon

“…Similarly, perfluorooctanoate (PFOA) and perfluorodecanoic acid (PFDA) elevate mitochondrial state 4 respiration and reduce state 3 respiration in isolated rat liver mitochondria (Keller et al, 1992; Langley, 1990) and are frequently detected in WWTP effluent. Finally, the general-use antimicrobial agent triclosan is a component of numerous personal care products and is a potent mitochondrial uncoupler in rat and human mast cells, primary human keratinocytes (Weatherly et al, 2016), and in 24-hour post-fertilization zebrafish embryos (Shim et al, 2016). Although the aforementioned mitochondrial effects were mostly described in mammalian laboratory models, a recent study of 12 diverse fish species demonstrated that 65–86% of human drug targets were evolutionarily conserved in the studied fish species, suggesting that the mechanisms of action of many CECs may share commonalities with fish (Brown et al, 2014).…”