The essential oils component p-cymene induces proton leak through Fo-ATP synthase and uncoupling of mitochondrial respiration

Abstract

Essential oils can be used as antimicrobial, antioxidant, and anticarcinogenic agents or to preserve and give flavors to foods. The activity of phenolic-rich essential oils has been observed in fractions containing thymol and carvacrol which show synergistic effects with their precursor p-cymene. Their mode of action is related to several targets in the cell but specific mechanisms of activity and cytotoxic effects remain poorly characterized. Given the importance of mitochondria for cellular functions and their critical role in a vast number of diseases, this work evaluated the effects of p-cymene on mitochondrial functions. It was observed that p-cymene did not change the oxygen consumption by respiratory chain (state 2 respiration). However, p-cymene decreased the mitochondrial membrane potential (Δψ), depressed the rate of ADP phosphorylation (state 3), and stimulated the oxygen consumption after phosphorylation of ADP (state 4). The respiratory control ratio (state 3/state 4) was decreased as a consequence of the inhibition of state 3 and stimulation of state 4 respiration but the ADP/O index remained unaltered as well as the mitochondrial Ca(2+) fluxes. Moreover, p-cymene did not induce mitochondrial membrane disruption but depressed the Δψ, and the stimulatory effect observed on state 4, similar to the effect observed on state 2 respiration plus ATP, was inhibited by oligomycin. These effects suggest that p-cymene allows a proton leak through the Fo fraction of the phosphorylative system, changing the mitochondrial proton motive force and ATP synthesis capacity. Therefore, these data suggest mitochondria as a target for p-cymene toxicity action mechanisms.

Keywords: ATP synthase; antimicrobial; essential oils; food additive; mitochondrial respiration; p-cymene; proton leak; uncoupling.

Figure 1

Chemical structure of p-cymene (1-isopropyl-4-methylbenzene).

Figure 2

Effect of p-cymene (PCy) on the respiratory states 2 (A), 3 (B) and 4 (C) of liver mitochondria induced by glutamate/malate. Mitochondrial respiration rates were determined by O₂ consumption using a Clark-type electrode. Notes: The data represent the means ± SD of 6 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 vs control.

Figure 3

Effect of p-cymene (PCy) on respiratory control ratio (RCR) (A) and ADP/O ratio (B). Notes: Indices were determined as described in Materials and methods and values are expressed as means ± SD of 6 independent experiments. **P < 0.01; ***P < 0.001 vs control.

Figure 4

Effects of p-cymene (PCy) on mitochondrial swelling. The traces, obtained by following the light scattering at 540 nm, are typical of 4 separate experiments with different mitochondrial preparations.

Figure 5

Effect of p-cymene (PCy) on state 2 plus ATP (A) and on state 4 respiration in the presence of oligomycin (st4-olig) (B). State 2 respiration was determined after energization of liver mitochondria with glutamate/malate and state 4 respiration after phosphorylation of ADP. State 4 respiration in the presence of oligomycin (st4-olig) was induced by the addition of ADP and oligomycin after the steady state of oxygen consumption characteristic of state 4 respiration had been reached. Notes: The data shown represent the means ± SD of 6 independent experiments. *P < 0.05; **P < 0.01 vs control; ⁺P < 0.05; ⁺⁺P < 0.01; ⁺⁺⁺P < 0.001 vs state 4 in the absence of oligomycin (st4).

Figure 6

Effect of p-cymene (PCy) on Ca²⁺ fluxes of mitochondria isolated from rat liver. Freshly isolated mitochondria were incubated at 0.25 mg/mL under standard conditions as described in the Materials and Methods section. Standard medium was supplemented with different concentrations of Ca²⁺ 1 minute before mitochondria energization with 5 mM succinate. Cyclosporin A (CsA) (0.85 μM) or p-cymene (25 nmol/mg protein) were added to the reaction medium 3 minutes prior to Ca²⁺ addition. The traces are typical of 6 independent experiments.