Endogenous Reactive Oxygen Species Is an Important Mediator of Miconazole Antifungal Effect

Abstract

We investigated the significance of endogenous reactive oxygen species (ROS) produced by fungi treated with miconazole. ROS production in Candida albicans was measured by a real-time fluorogenic assay. The level of ROS production was increased by miconazole at the MIC (0.125 g/ml) and was enhanced further in a dose-dependent manner, with a fourfold increase detected when miconazole was used at 12.5 g/ml. This increase in the level of ROS production was completely inhibited by pyrrolidinedithiocarbamate (PDTC), an antioxidant, at 10 M. In a colony formation assay, the decrease in cell viability associated with miconazole treatment was significantly prevented by addition of PDTC. Moreover, the level of ROS production by 10 clinical isolates of Candida species was inversely correlated with the miconazole MIC (r ‫؍‬ ؊0.8818; P < 0.01). These results indicate that ROS production is important to the antifungal activity of miconazole.Candidiasis is a life-threatening disease in patients with immune suppression. Azoles are used widely for the treatment of candidiasis, but some clinical isolates show resistance (11,28). Further investigation of the mechanisms underlying the antifungal effects of azoles may aid in the development of new treatment strategies.Azoles exert a cytostatic or cytotoxic effect via inhibition of synthesis reactions in the metabolic pathways of essential fungal cell membrane components including ergosterol (29,30). Their primary target is the cytochrome P450-catalyzed 14␣-demethylation of ergosterol precursors. Mutations or overexpression of 14␣-demethylase, encoded by the ERG11 gene, as well as changes in the ergosterol synthesis pathway reportedly participates in the induction of resistance (14,19,20,25).Since it was found that miconazole, ketoconazole, and deacetylated ketoconazole were inserted in a lipid layer (6) and miconazole induced the release of K ϩ and intracellular ATP from Candida species (3, 4, 7), these agents may cause direct membrane damage.Recent studies clarified that a decrease in drug concentration brought about by energy-dependent efflux pumps is usually involved in multidrug resistance including azole resistance (1,2,16,26). This pumping system closely resembles a system in cancer cells based on the P-glycoprotein encoded by the MDR-1 gene. Thus, common biologic mechanisms shared with other eukaryotic cells are involved in the antifungal effects of azoles.In eukaryotic cells, mitochondria are common organelles that represent an important source of reactive oxygen species (ROS). Many cellular stresses such as irradiation and cytotoxic drugs cause growth inhibition and the death of mammalian cells via endogenous ROS production (5, 15, 27). ROS produced by granulocytes or monocytes are known to exert activity against fungi (9, 31). Furthermore, recent studies demonstrated that Candida albicans possesses an ROS scavenger, superoxide dismutase; this suggests that fungi may require a cytoprotective mechanism against not only exogenous ROS but also endogenous, fungus-derived ROS (1...

Miconazole Induces Changes in Actin Cytoskeleton prior to Reactive Oxygen Species Induction in Yeast

“…1 Supported by a postdoctoral fellowship from Katholieke Universiteit Leuven (Industrial Research Fellow). 2 A senior non-clinical fellow of the Medical Research Council UK (G117/394). 3 Supported by a predoctoral fellowship from IWT-Vlaanderen.…”

“…A similar approach was previously used by Parsons et al (4) to uncover yeast tolerance mechanisms against various chemical compounds, including the triazole fluconazole. However, miconazole and fluconazole are characterized by different activity features: fluconazole does not seem to induce ROS to the same extent as miconazole in susceptible fungi (2,3).…”

“…Recently, an additional mode of antifungal action was uncovered for miconazole. Apart from inhibition of ergosterol biosynthesis, miconazole induces reactive oxygen species (ROS) 6 in susceptible fungi, leading to fungal cell death (2,3).…”

Trehalose Is an Important Mediator of Cap1p Oxidative Stress Response in Candida albicans

“…3) Moreover, ROS generation is involved in the mechanisms of many antifungal agents. [4][5][6] Oxidative stress could therefore be a frequent challenge for C. albicans to survive and cause diseases in its host.…”

CaIPF7817 is involved in the regulation of redox homeostasis in Candida albicans

“…Intracellular levels of ROS were measured with DCFH-DA (2 0 ,7 0 -dichlorodihydro-fluorescein diacetate, Molecular Probes, USA) [18]. Briefly, C. albicans cells cultured in YPD medium at the exponential growth phase (OD 600 = 0.5) were collected by centrifugation (3000g, 5 min, 4°C) and washed three times with PBS.…”