Cytoprotective Effects of Melatonin Against Amitriptyline-Induced Toxicity in Isolated Rat Hepatocytes

© 2015 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2015, 5(3), 329-334 doi: 10.15171/apb.2015.046 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
Melatonin is produced in the vertebrate pineal gland, the retina and possibly some other organs.This hormone plays several physiological roles such as regulation of sleep.Also there are many reports that melatonin's antioxidant action contributes to the protection of the organism from carcinogenesis and neurodegenerative disorders.2][3][4] There are many reports on melatonin's protective effects against different chemicals-induced hepatotoxicity. 5,6t has been reported that amitriptyline increased liver enzymes in patients that received it continuously. 7][10] It was previously reported that amitriptyline induced oxidative stress, it increased ROS formation and lipid peroxidation also amitriptyline increased GSH consumption in isolated rat hepatocytes. 9In addition, a previous study showed that amitriptyline caused mitochondrial membrane potential reduction. 9Based on our medical article data bases, there are few studies on the hepatoprotective role of antioxidants against amitriptyline-induced hepatotoxicity.In the present study, we investigated, for the first time, the protective roles of melatonin against cytotoxicity induced by amitriptyline towards freshly isolated rat hepatocytes.Trypan blue exclusion test was used to measuring the percent of viable cells.The possibility of reactive oxygen species formation and lipid peroxidation was assessed and the effect of amitriptyline on intracellular glutathione was evaluated.In addition, the effect of amitriptylin on hepatocyte mitochondria was studied.

Chemicals
Amitriptyline, melatonin (N-Acetyl-5methoxytryptamine) and Collagenase were obtained Sigma Aldrich chemical Co (St. Louis, USA).Other reagents were purchased from Merck chemical CO (Darmstadt, Germany).Amitriptyline was dissolved in water and melatonin was dissolved in methanol.The amount of solvent in media was less than 20µl.The solvents had no effects on toxin or antioxidant effects.Male Sprague-Dawley rats (250-300 g) were housed in ventilated plastic cages with 12h light photoperiod and an environmental temperature of 21-23°C with a 50-60% relative humidity.Animals were fed a normal chow diet and water ad libitum.The animals were handled and used according the animal handling protocol that approved by a local ethic committee in Tabriz University of medical sciences, Tabriz, Iran.The rat liver was perfused with different buffer solutions through the portal vein.Hepatocytes were isolated from liver using collagenase perfusion technique which has been described in details before by Eghbal et al. 9 The collagenase enzyme in buffer solution destructed the liver tissue and lead to easily isolated of hepatocytes during the next steps. 9Isolated hepatocytes (10 mL, 10 6 cells/mL) were suspended in the Krebs-Henseleit buffer (pH=7.4) in continuously rotating round bottom flasks, under an atmosphere of carbogen gas ( 95% O 2 and 5% CO 2 ) in a 37 °C water bath.Only the cells with viability of over the 85% were used.

Lipid peroxidation Measurement
Hepatocytes lipid peroxidation was detected by measuring of thiobarbituric acid reactive substance (TBARS) that formed during the decomposition of lipid hydro peroxides.250µl of trichloroacetic acid (TCA, 70%w/v) was added to 1ml of hepatocytes suspension (10 6 cell) and centrifuged at 3000g for 15 min, then 1 ml of thiobarbituric acid (0.8%w/v) added to supernatant and boiled for 20 min.The absorbance was measured at 532nm in an Ultrospec 2000 spectrophotometer. 21,22tochondrial membrane potential Mitochondrial membrane potential was evaluated as a marker of toxicity induced by amitriptyline.To determining mitochondrial membrane potential Rhodamine 123 (the fluorescent dye) was used.This dye accumulates in intact mitochondria by facilitated diffusion.When the mitochondrial membrane potential is reduced by a toxin the amount of Rhodamine 123 in media is increased.One mL sample was taken from the cell suspension at programmed time points, and centrifuged at 1000 rpm for 1 minute.The cell was resuspensed in 2 ml of krebs-Henseleit buffer containing 1.5 µM Rhodamine 123 and incubated at 37°C water bath for 10 minutes.Hepatocytes were separated by centrifugation (3000 rpm for one minute) and the amount of rhodamine 123 appearing in the incubation medium were determined fluorimeterically at 490 nm excitation and 520 nm emission wavelengths using a Jasco® FP-750 spectrofluorometer.1,23

Determination of Hepatocytes GSH/GSSG content
To determine the hepatocyte glutathione (GSH) content the method of Ellman was used. 24A 1 ml aliquot of the cell suspension (10 6 cells) was taken and 2 ml of 5% TCA was added and centrifuged.Then 0.5 ml of Ellman's reagent (0.0198% DTNB in 1% sodium citrate) and 3 ml of the phosphate buffer (pH 8.0) were added.The absorbance of the developed color was measured at 412 nm using a Biotech Pharmacia Ultrospec ® 2000 spectro-photometer.Cell samples were reduced with potassium borohydride (KBH4) to prevent GSH oxidation during the experiment. 25The enzymatic recycling method was used to assess the hepatocyte oxidized glutathione (GSSG) level. 26Cellular GSH content was covalently bonded to 2-vinylpyridine at first.Then the excess 2-vinylpyridine was neutralized with thriethanolamine, and GSSG was reduced to GSH using the glutathione reductase enzyme and NADPH.The amount of GSH formed was measured as already described for GSH using the Ellman reagent (0.0198% DTNB in 1% sodium citrate). 2,27

Statistical analysis
Results are given as the Mean±SEM for at least three independent experiments.Statistical analysis for the control and experimental groups was performed by a one-way ANOVA (analysis of variance) test followed by a Tukey's post hoc test.A P < 0.05 was considered as a significant difference.

Results
Trypan blue exclusion test was used to determine the ability of hepatocytes to maintain their viability with amitriptyline alone or in combination with melatonin.Previous finding showed that amitriptyline caused cell death in a concentration dependent manner.The concentration in which amitriptyline caused 50 % cell death (LC 50 ) was found to be 40µM. 9Our findings indicated that an effective dose of melatonin that provided suitable protection was 1mM (Table 1).Melatonin administration effectively prevented cell death induced by amitriptyline (P<0.05)(Table 1).Markers such as ROS formation, lipid peroxidation, cellular glutathione content, and mitochondrial membrane potential were measured to study the    Previous findings showed that a notable amount of thiobarbituric acid reactive substances (TBARS) was formed in amitriptyline-treated rat hepatocytes as compared to the control group. 9This indicates lipid peroxidation induced by cytotoxic concentrations of the drug.As shown in Figure 2 incubation of hepatocytes with 1mM of melatonin, prevented lipid peroxidation induced by amitriptyline (P<0.05).Previous findings showed that the LC 50 dose of amitriptyline (40 µM) caused a reduction in mitochondrial membrane potential as measured by rhodamine 123 test. 9This indicates that mitochondria could be a target for amitriptyline. 9Melatonin was administered with amitriptyline to investigate if it could alleviate mitochondrial injury induced by amitriptyline.In comparison between the groups receiving amitriptyline alone and the group receiving amitriptyline in combination with 1mM melatonin, the degree of permeability of Rhodamine to the mitochondria was increased by melatonin significantly.It was found that melatonin effectively prevented mitochondrial depolarization caused by amitriptyline (Table 2).Previous findings indicated that hepatocytes glutathione content was diminished notably when hepatocytes were treated with amitriptyline (40µm). 9According to Table 3, comparing the group receiving only amitriptyline to the group receiving amitriptyline and 1mM melatonin, GSH content of cells was conspicuously increased in the melatonin-supplemented groups (P<0.05).According to previous results, the amount of oxidized glutathione (GSSG) was increased in hepatocytes treated with 40µm amitriptyline. 9According to Table 4, GSSG levels of amitriptyline-exposed cells were notably diminished in the melatonin-supplemented groups (P<0.05).

Discussion
We have recently demonstrated that the cytotoxicity induced by amitriptyline was accompanied with ROS formation, lipid peroxidation, and mitochondrial depolarization.Moreover, reduction in cellular glutathione content was occurred.These results confirm the existence of the process of oxidative stress in hepatotoxicity due to amitriptyline. 9 Present results showed that hepatocytes supplementation with 1mM melatonin reduced cell death induced by amitriptyline and diminished the consequences of amitriptyline induced toxicity such as ROS generation, TBARS production and mitochondrial collapse.The protective effects of melatonin against amitriptyline could be due to its capability in scavenging reactive metabolites and conjugating with them.These findings are in line with the previous studies regarding the radical scavenger effects of melatonin. 3,4Melatonin can rise the glutathione saving and decreases GSSG formation during amitriptyline toxicity notably (Table 3, 4).This might indicate the importance of melatonin in alleviating oxidative stress and scavenging reactive species to prevent the glutathione consumption as one of the main cellular defense mechanisms against toxic insults.This finding also supports the previous studies regarding the antioxidant effects of melatonin. 3,4Mitochondrial depolarization can cause energy crisis and releasing of apoptotic signaling molecules, which could finally encounter cell death. 27Administrations of 1mM melatonin provide protection against amitriptyline-induced mitochondrial collapse.There may be other functions of melatonin, yet undiscovered, which enhance its ability to protect against molecular damage by oxygen and toxic reactants.Protective effect of melatonin against amitriptyline-induced mitochondrial damage is through its activity in attenuating oxidative stress and scavenging reactive species.

Conclusion
According to our results, it could be concluded that melatonin is an effective protective agent in preventing amitriptyline-induced hepatotoxicity.We recommend further clinical trial studies on the antioxidant effects of melatonin in patients receiving amitriptyline.Also, the protective effect of melatonin against amitriptyline-induced toxicity proposes this agent as the subject of further studies for preventing different xenobiotics-induced liver damages, especially those accompanied by oxidative stress.

Figure 1 .
Figure 1.Protective effect of melatonin against amitriptyline induced ROS formation in isolated rat hepatocytes Isolated rat hepatocytes (10 6 cells/mL) were incubated at 37 °C in rotating round bottom flasks with 95 % O2 and 5 % CO2 in Krebs-Henseleit buffer (pH 7.4).The results shown represent Mean ± SE for three separate experiments.*: Significantly different as compared to control group (P<0.05).**: Significantly different from amitriptyline-treated group (P<0.05)

Table 1 .
Protective effect of melatonin against amitriptyline induced toxicity in isolated rat hepatocytes

Table 2 .
protective effect of melatonin on mitochondrial toxicity induced by amitriptyline

Table 3 .
Effect of melatonin on GSH consumption in presence of amitriptyline

Table 4 .
Effect of melatonin on GSSG level in presence of amitriptyline Isolated rat hepatocytes (10 6 cells/mL) were incubated at 37°C in rotating round bottom flasks with 95 % O2 and 5 % CO2 in Krebs-Henseleit buffer (pH 7.4).GSSG level was measured as described by Irfan Rahman et al (2006) The results shown represent Mean±SE for three separate experiments.*: Significantly different as compared to control group (P<0.05).