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The aims of this study were: (a) to assess whether the increased oxidative stress in otitis media with effusion (OME) induced in guinea pigs by histamine injection into the middle ear cavity is reflected by lipid peroxidation in erythrocytes, plasma, and middle ear effusion fluid; (b) to survey the alterations of oxidant and antioxidant enzyme activities in experimental OME; and (c) to determine the effects of melatonin and methylprednisolone on this oxidative stress. Malondialdehyde (MDA) level, erythrocyte total (enzymatic plus non-enzymatic) superoxide scavenger activity (TSSA), non-enzymatic superoxide scavenger activity (NSSA), superoxide dismutase (SOD), catalase (CAT), and xanthine oxidase (XO) activities were measured in 4 groups of 7 guinea pigs at 3 hr after injection of 0.1 ml of histamine (or saline) into the middle ear. Group I was the control group, Group II was an experimental group with OME induced by histamine, Group III was a melatonin-pretreated OME group, and Group IV was a methylprednisolone-pretreated OME group. In erythrocyte, plasma, and middle ear effusion samples, MDA levels were significantly increased in guinea pigs with OME (Group II), compared to controls (Group I); erythrocyte TSSA and SOD activities were lower and erythrocyte XO activity was increased in guinea pigs with OME (Group II) compared to controls (Group I). No significant differences were found in erythrocyte NSSA and CAT activities. In Group III, pretreatment of guinea pigs with ip melatonin at 1 hr prior to histamine induction of OME decreased the erythrocyte, plasma, and effusion MDA levels, compared to Group II; erythrocyte XO activity was diminished and erythrocyte TSSA, SOD, and CAT activities were increased in Group III compared to Group II. In Group IV, pretreatment of guinea pigs with ip methylprednisolone at 1 hr prior to histamine induction of OME decreased the plasma and effusion MDA levels and increased the erythrocyte TSSA and SOD activities, compared to Group II. These results suggest that reactive oxygen species (ROS) play a role in histamine-induced OME. Pretreatment with ip melatonin or methylprednisolone both decrease the ROS generated by experimental OME, but melatonin appears to be more effective than methylprednisolone.