Mitochondrial toxicity of triclosan on mammalian cells

Abstract

Effects of triclosan (5-chloro-2'-(2,4-dichlorophenoxy)phenol) on mammalian cells were investigated using human peripheral blood mono nuclear cells (PBMC), keratinocytes (HaCaT), porcine spermatozoa and kidney tubular epithelial cells (PK-15), murine pancreatic islets (MIN-6) and neuroblastoma cells (MNA) as targets. We show that triclosan (1-10 μg ml^-1) depolarised the mitochondria, upshifted the rate of glucose consumption in PMBC, HaCaT, PK-15 and MNA, and subsequently induced metabolic acidosis. Triclosan induced a regression of insulin producing pancreatic islets into tiny pycnotic cells and necrotic death. Short exposure to low concentrations of triclosan (30 min, ≤1 μg/ml) paralyzed the high amplitude tail beating and progressive motility of spermatozoa, within 30 min exposure, depolarized the spermatozoan mitochondria and hyperpolarised the acrosome region of the sperm head and the flagellar fibrous sheath (distal part of the flagellum). Experiments with isolated rat liver mitochondria showed that triclosan impaired oxidative phosphorylation, downshifted ATP synthesis, uncoupled respiration and provoked excessive oxygen uptake. These exposure concentrations are 100-1000 fold lower that those permitted in consumer goods. The mitochondriotoxic mechanism of triclosan differs from that of valinomycin, cereulide and the enniatins by not involving potassium ionophoric activity.

Keywords: Acidosis; BCF, bioconcentration factor; EC50, concentration that diminishes the respective vitality parameter by ≥50%; Electric transmembrane potential; Glycolysis; HaCaT, a spontaneously immortalized (non-neoplastic) keratinocyte cell line; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide; MIC, minimal inhibitory concentration; MIN-6, a murine pancreatic beta cell line; MNA, a murine neuroblastoma cells; Oxidative phosphorylation; PBMC, monocyte-enriched peripheral blood mononuclear cells; PI, propidium iodide; PK-15, a porcine kidney tubular epithelial cell line; PN, pyridine nucleotides; RLM, rat liver mitochondria; Sperm motility; TPP+, tetraphenylphosphonium; Uncoupler; ΔΨ, electric transmembrane potential; ΔΨm, membrane potential of the mitochondrial membrane; ΔΨp, membrane potential of the plasma membrane.

Fig. 1

Effects of triclosan on glucose metabolism by human primary PBMC. PBMC freshly isolated from healthy human donor blood were exposed to 1–100 μg/ml of triclosan for 44 h. Concentration of glucose in the medium was measured at indicated time points during the 44 h exposure (Panel A). The resazurin fluorescence as an indicator of the presence of NADH and pH in the medium an indicator of anaerobic metabolism were measured at the end of the exposure (at 44 h) (Panel B).

Fig. 2

Exposure to triclosan dissipated mitochondrial membrane potential in human PBMC and keratinocytes (HaCaT), porcine kidney tubular epithelial cells (PK-15) and spermatozoa, murine neuroblastoma cells (MNA) and pancreatic islets (MIN-6, insulin producing). After exposure to the indicated concentrations (μg/ml) and times (minutes, hours) of triclosan, the cells were stained with the membrane potential responsive fluorogenic dye JC-1 (μg/ml) or double stained with (JC-1 and propidium iodide, PI; PBMC and HaCaT). The fluorescent emission of JC-1 shifts from red-orange via yellow to green when the membrane potential (ΔΨ) diminishes from ≥150 mV toward ≤100 mV. Necrotic cells (PI positive) are visible in PBMC (30 μg) as swollen, intensely red cells. The images are representative of three independent microscopic views. Scale bar, 30 μm.

Fig. 3

Microscopic assessment of death of human, porcine and murine cells upon exposure to triclosan. The concentration of triclosan exposure (24 h) is shown for each panel. After exposure, the cells were double stained with acetoxymethyl calcein (live stain, green fluorescing) and propidium iodide (PI, death stain, red fluorescence indicates permeabilisation of the cell). The cells exposed to vehicle only (methanol) show no permeability to PI. The cells exposed to triclosan show dose-dependent increase of PI-permeabilized (red) cells. At the highest exposures, the cytoplasmic content (green) has leaked out, leaving the red stained cell nuclei visible. Measure bar, 30 μm.

Fig. 4

(A) Original flow cytometric dot plots of spermatozoa exposed to 0.5–18 μg ml⁻¹ triclosan (dilution step = 2). Top row: SYBR14 (left panel – viable)/PI (right panel – dead) stained. Middle row: top panel JC-1 aggregates (ΔΨ high), bottom panel JC-1 monomers (ΔΨ lost). Bottom row: M540 (bottom panels)/To-Pro3 (top panel – dead). M540 bottom left panel: viable intact. M540 bottom right panel: membrane disordered. (B) Quantitative effects of triclosan on selected viability parameters of boar spermatozoa measured by flowcytometry. SYBR14/PI, M540/To-Pro3, JC-1 aggregation value measured after 24 h exposure at 20 °C.

Fig. 5

Effects of triclosan on the membrane potential, redox state of pyridine nucleotides and on oxidative phosphorylation in isolated rat liver mitochondria. ΔΨ_m was measured with TPP⁺-selective electrode in rat liver mitochondria (RLM) (1 mg protein ml⁻¹) in standard medium with 5 mM glutamate plus 5 mM malate as the respiring substrates. Triclosan (TCS) (A) or FCCP (B) was added at concentration and time points indicated. Panel A shows TCS induced a concentration-dependent decrease of the mitochondrial membrane potential (ΔΨ_m, measured with TPP⁺-selective electrode) of isolated RLM. Panel B shows FCCP induced a concentration-dependent decrease of the mitochondrial membrane potential. Panel C shows TCS induced a concentration-dependent decrease of pyridine nucleotides (PN) fluorescence and uncoupling of oxidative phosphorylation in RLM. Trace 0 shows the vehicle (methanol) control. The final concentrations of ADP and FCCP were 200 μM and 1 μM, respectively. The traces shown are representative for three separate experiments.

Fig. 6

The effect of triclosan (TCS) and 2,4-dinitrophenol (DNP) on rates of oxygen consumption and oxidative phosphorylation of RLM (1 mg protein ml⁻¹ in standard medium) respiring on 5 mM glutamate plus 5 mM malate (A) or succinate in the presence of rotenone (B). Trace 0 shows the vehicle (methanol) control. The final concentrations of ADP and DNP were 200 μM and 50 μM, respectively. The traces shown are representative for three separate experiments.