Melatonin receptor antagonist luzindole is a powerful radical scavenger in vitro

To the Editor The radical scavenging potential of melatonin is well documented [1]. Consequentially, exogenous administration of melatonin has proven useful in different models of stress in vivo, such as in sepsis [2], after ischemia/reperfusion [3, 4], and in a variety of toxic challenges [5, 6]. Some of these beneficial effects of melatonin are mediated via receptor dependent processes [7]. To identify the relevance of melatonin receptors, the receptor antagonist luzindole was introduced. This substance displays an affinity to all melatonin receptor (MT) subtypes, MT1, MT2, and MT3 (quinone reductase 2) [8]. Despite the extensive use of luzindole, many pharmacological aspects of this substance remain unknown. Possible dose-dependent variations in the response to luzidole have been reported [9], partial agonist behavior was suggested in human leukemia cells [10], and only limited data are available with respect to precise agonist–antagonist relationships [11]. The failure of luzindole to antagonize melatonin s effect on hepatocellular redox state in a recent study on hemorrhagic shock [12] prompted the question on possible antioxidant properties of luzindole itself. Therefore, this study was designed to evaluate the radical scavenging properties of melatonin receptor antagonist luzindole in vitro. Radical scavenging was evaluated using a spectrophotometrical scavenger competition assay based on the formation of 2,2¢-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical cation [13]. All measurements were carried out at room temperature and performed in triplicates. Total antioxidant capacity was determined for luzindole, melatonin, ascorbic acid, and vehicle dimethyl sulfoxide (DMSO). ABTS was diluted in water to a concentration of 7.0 mm and incubated with potassium persulfate (2.45 mm final concentration) for 12 hr at room temperature, protected from light, for formation of ABTS radical cation (ABTS). ABTS was diluted with 5 mm phosphate buffered saline (PBS), pH 7.2, to an absorbance value of 0.7 at 734 nm in a spectrophotometer (Ultrospect2000; Pharmacia Biotech, Piscataway, NJ, USA). Luzindole, melatonin, and ascorbic acid were freshly dissolved and added to ABTS (final concentrations: 5.0 lm, 10.0 lm, and 50.0 lm for each substance). Total antioxidant capacity is expressed as the percentage of inhibition of the absorbance of ABTS at room temperature at 734 nm as a function of time. A strong inhibition of ABTS radical was achieved for melatonin, luzindole, and ascorbic acid within 30 min, using the concentration of 5.0 lm (Fig. 1A) and 10.0 lm (Fig. 1B). No significant inhibition of ABTSwas detected for DMSO. At a concentration of 50 lm, melatonin, luzindole, and ascorbic acid reached an inhibition of ABTS >90%within seconds, while DMSO did not affect the absorption of ABTS for 30 min (data not shown). These data demonstrate that luzindole is a potent antioxidant itself, exhibiting an inhibition of ABTS radical of almost 80% at a concentration of 10 lm after 30 min in vitro. The ABTS assay was previously shown to be a reliable method to estimate the antioxidant capacity of a variety of substances, including food products and indoles [13, 14]. The results obtained for melatonin and ascorbic