PURPOSE: Smoking is a risk factor in the development of a variety of neuroretinal diseases. Therefore, we have investigated the effects of hydroquinone (HQ), a toxicant that is present in high concentrations in cigarette smoke, on a human retinal Müller cell line (MIO-M1). METHODS: MIO-M1 cells were treated for 24h with four different concentrations of HQ (200μM, 100μM, 50μM, and 25μM). Assays were used to measure cell viability, reactive oxygen/nitrogen species (ROS/RNS), mitochondrial dehydrogenase activity (WST assay), caspase-3/7 activity and lactate dehydrogenase (LDH) levels. Western blot analyses with anti-LC3 and anti-GAPDH antibodies were performed on HQ-treated samples. Some cultures were treated with 4μM rapamycin, to induce autophagy, with and without the autophagy inhibitor 3-methyl-adenine (3MA), and levels of ROS/RNS and LDH were measured. RESULTS: Our findings show that HQ reduced cell viability at four different concentrations tested (200, 100, 50 and 25μM); decreased mitochondrial function at concentrations of 200 and 100μM; increased ROS/RNS activity at all the concentrations tested and increased LDH levels at concentrations of 200, 100 and 50μM. Caspase-3/7 activities were not modified by HQ. However, treatment of these cells with this agent resulted in the appearance of the autophagy associated LC3-II band. Pre-treatment with 3MA reduced the ROS/RNS and LDH levels of the HQ-treated and rapamycin-treated cells. CONCLUSION: Our study suggests that HQ damages the MIO-M1 cells through oxidative, mitochondrial and autophagic pathways and not caspase-related apoptosis.
PURPOSE: Smoking is a risk factor in the development of a variety of neuroretinal diseases. Therefore, we have investigated the effects of hydroquinone (HQ), a toxicant that is present in high concentrations in cigarette smoke, on a human retinal Müller cell line (MIO-M1). METHODS: MIO-M1 cells were treated for 24h with four different concentrations of HQ (200μM, 100μM, 50μM, and 25μM). Assays were used to measure cell viability, reactive oxygen/nitrogen species (ROS/RNS), mitochondrial dehydrogenase activity (WST assay), caspase-3/7 activity and lactate dehydrogenase (LDH) levels. Western blot analyses with anti-LC3 and anti-GAPDH antibodies were performed on HQ-treated samples. Some cultures were treated with 4μM rapamycin, to induce autophagy, with and without the autophagy inhibitor 3-methyl-adenine (3MA), and levels of ROS/RNS and LDH were measured. RESULTS: Our findings show that HQ reduced cell viability at four different concentrations tested (200, 100, 50 and 25μM); decreased mitochondrial function at concentrations of 200 and 100μM; increased ROS/RNS activity at all the concentrations tested and increased LDH levels at concentrations of 200, 100 and 50μM. Caspase-3/7 activities were not modified by HQ. However, treatment of these cells with this agent resulted in the appearance of the autophagy associated LC3-II band. Pre-treatment with 3MA reduced the ROS/RNS and LDH levels of the HQ-treated and rapamycin-treated cells. CONCLUSION: Our study suggests that HQ damages the MIO-M1 cells through oxidative, mitochondrial and autophagic pathways and not caspase-related apoptosis.
Authors: Claudio Ramírez; Javier Cáceres-del-Carpio; Justin Chu; Joshua Chu; M Tarek Moustafa; Marilyn Chwa; G Astrid Limb; Baruch D Kuppermann; M Cristina Kenney Journal: J Ocul Pharmacol Ther Date: 2015-12-01 Impact factor: 2.671