Protective Roles of N-acetyl Cysteine and/or Taurine against Sumatriptan-Induced Hepatotoxicity

2016 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, 2016, 6(4), 627-637 doi: 10.15171/apb.2016.077 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
Primary headache disorder migraine is the 3rd disabling disease in the world and affects more than 10 % of people worldwide. 1 It has been estimated that more than thirty million people in the United States suffer from migraine. 2 Triptans are a drugs class with proven effect in acute treatment of migraine attacks. The first member of this class of drugs sumatriptan was presented to the market within several formulations. Clearance of organic anions and bile acids from the liver may be affected by sumatriptan. 3,4 The major route of elimination of sumatriptan is metabolism in the liver. 5 This medication undergoes hepatic metabolic first-pass effects. 6 Triptans have various bioavailability and half-life. Oral bioavailability of sumatriptan is low because of first pass metabolism. 7,8 Sumatriptan pharmacokinetics is affected by CYP 3A4 inhibitors. 9 Sumatriptan is also metabolized by monoamine oxidase enzyme. 10 Some studies have suggested that there is little information about triptans poisoning. Toxic doses of triptans could vary depending on some circumstances such as pregnancy. 11 On the base of Germany poisons information center, in total, fifty nine cases of triptans' overdose have been registered. Children constitute high percentages of the patients (forty two cases of al). 12 Moreover, ischemic colitis thought to be related to sumatriptan for migraines. 13 Moreover there are case reports of adverse effect of triptans on kidney e.g. subacute ischemic injuries of the kidney or renal infarction. 14,15 Most recent toxic effects of triptans on liver and the cases of hepatotoxicity of triptans have been reported. 16 Another case report was a 17-year-old girl who developed hepatotoxicity during treatment with antimigraine triptans. 17 Sumatriptan is in the possible hepatotoxic class of drugs according to its structural moiety. 18 In vitro studies performed in tube in absence of cells, suggested that sumatriptan has direct scavenging activity on free radicals, 19 but this medication have several metabolites after enzymatic processes in the body, thus, the overall outcome should be clarified. Water-soluble ROS scavenger N-acetylcysteine (NAC) is naturally formed in garlic and onion. 20 This neuroprotective agent is a precursor of the glutathione (GSH) and could interact directly with ROS. 21 NAC is anti-inflammatory agent and displays beneficial effects on toxicity induced by HMG-CoA reductase inhibitors, arsenic and CCl 4 . 22-25 2-aminoethanesulfonic acid (taurine) is a cell membrane stabilizer and has shown protective effects against toxicity of antiseizures such as phenytoin and carbamazepine in several organs of the body including testes brain, liver as well as retina. [26][27][28][29] Beneficial effects of natural flavonol quercetin have been described in cardiovascular disorders, hepatotoxicity and cancer 30,31 as well as radiotoxicity. 32 This glycoside reduces hepatotoxicity included by sodium fluoride and acetaminophen. 33-35 Lipophilic ROS scavenger αtocopherol (vitamin E) is a supplementary nutrition witch has displayed benefical effects on carcinogenic effects of chemicals specially in combination with sellenium. 36 Oxidative stress induced by thallium and mood stabilizers such as valproic acid in hepatocytes has been effectively reduced by vitamin E. 37,38 The exact mechanisms of harmful effects of sumatriptan in hepatocytes have not yet been illustrated. The major aim of this work was to determine the cellular mechanisms of harmful effects of sumatriptan in freshly isolated rat parenchymal hepatocytes and to explore the beneficial roles of NAC, taurine, quercetin and/or αtocopherol.

Materials
The materials used in the present study were pure and prepared from Sigma-Aldrich Co. (Taufkirchen, Germany). Becoming stable hepatocytes had been preincubated for 30 min before addition of test materials. 1bromoalkanes have been employed to deplete hepatocytes glutathione. 39

Animals
Male Albino rats of Sprague-Dawley strain (250 -320 g) had been acquired from Medical Sciences University of Tabriz (Tabriz, Iran). Separate plastic cages were employed to keep animals under standard diet of chow and water (ad-lib) with controlled temperature (21 °C -23 °C). All animals were exposed to photoperiod of light /dark 12:12 h. All tests were fulfilled under ethical standards determined by the local Committee of Animal Experimentation of Medical Sciences University of Tabriz.

Cell Preparation
Collagenase perfusion was performed to isolate hepatocytes as described previously. 40 Briefly after removal of Ca 2+ with chelator, digestive enzyme collagenase has been employed to prepare singlet and fresh parenchymal hepatocytes. To assess viability of the cells using trypan blue, equal portions of the test hepatocytes were taken at 60, 120 and 180 minutes after incubation. 40,41 In all experiments about 80-90 percent of the viable parenchymal hepatocytes were acquired under circulation of combination of 95 % oxygen and 5 % CO 2 atmosphere. In all experiments rat parenchymal liver cells have been suspended in Krebs-Henseleit buffer media with the concentration of 10 6 cells/ml, at pH 7.4 and 37°C in round-bottom flasks 30 min prior to the addition of sumatriptan (5 mM) and/or other materials. Avoiding very toxic circumstances, in this work, LC 50 concentrations of sumatriptan succinate (5 mM) after 2 h of incubation, have been calculated using dose-response curves based on a regression plot of three different concentrations. 42

Mitochondrial Membrane Potential (MMP) Assay
Rhodamine123 were used to assess the MMP. 44 The mitochondria uptake this dye and redistribution of rhodamine123 from injured mitochondria to the incubation medium could be measured spectroflourometrically (Excitation: 490 nm, Emission: 520 nm). A FP-750 Jasco fluorescence spectrophotometer (Tokyo, Japan) was used to determine the rhodamine123 levels. 45

Lysosomal Damage Assay
In the present study acridine orange were used to assess parenchymal hepatocyte lysosomal damage (Excitation: 495 nm, Emission: 530 nm). The lysosomes uptake this dye and redistribution of acridine orange from injured lysosomes to the medium could be measured using a FP-750 Jasco fluorescence spectrophotometer (Tokyo, Japan). 38

Lipid Peroxide Production Assay
Production of lipid peroxides has been measured by assessing the thiobarbituric acid reactive substances (TBARS). Absorbance at 530 nm was measured using UV spectrophotometer at several time intervals as previously described. 46

Reduced Glutathione Level Assay
Pure reduced glutathione was used as standards .To measure reduced glutathione by HPLC using a Bondapak NH2 column (Water Associates, Milford, MA) samples were deproteinized (by meta phosphoric acid 5 %) and then derivatized with dinitrofluorobenzene and iodoacetic acid as previous described. 47,48 Statistical Analysis These data have been compared by ANOVA statistical method followed by post hoc Tukey's test (with a p-value | 629

Hepatocytes Viability
Following incubation of hepatocytes for 3 hours (without any treatment), viability of the control cells was 85 %. In comparison to the control, membrane lysis significantly (p-value <0.05) increased in hepatocytes incubated with sumatriptan concentration-dependently. After 120 min incubation of hepatocytes with sumatriptan, the calculated LC 50 (i.e., 50 % membrane lysis within 120 min) was 5 mM (Table 1). This toxicity marker which measured by trypan blue dye exclusion test, was significantly reduced by taurine, NAC, quercetin as well as anti oxidants (BHT, Vitamin E) (p-value <0.05). Moreover, sumatriptan induced hepatocyte lysis has been prevented by l-glutamine (l-Gln) and fructose as ATP generators, cytochrome P450 enzyme inhibitors, endocytosis inhibitors chloroquine and methylamine as well as l-carnitine and trifluoperazine (TFP) as MPT pore sealants (Table 1). Our results showed that hepatocyte lysis notably increased in case of cytochrome P450 induction by pretreatment with phenobarbital for 3 days (Figure 1). In this circumstance hepatocyte lysis decreased by both CYP inhibitors cimetidine (2 mM) and 4-methylpyrazole (4-MP) (500 µM) (p-value < 0.05). Membrane lysis was not induced significantly (p-value < 0.05) by the protective agents and cytochrome P450 inhibitors as well as 1-bromoheptane at concentrations used when administered without sumatriptan (data not shown).

ROS Levels
A noticeable increase in ROS generation was observed in hepatocytes exposed to sumatriptan. ROS formation was significantly (p-value <0.05) reduced by incubation of the hepatocytes with taurine and quercetin as well as NAC. Also, ROS formation was significantly (p-value <0.05) reduced by treatment of isolated hepatocytes with aforementioned anti oxidants, l-Gln, fructose, cytochrome 450 inhibitors, l-carnitine, TFP as well as endocytosis inhibitors (Figure 2). Sumatriptan-induced ROS levels was in turn increased by depleting hepatocyte GSH with 1-bromoalkane, demonstrating the impact of glutathione in high ROS levels induced by sumatriptan ( Figure 2).

MMP
MMP was reduced by sumatriptan administration to the cells compared to the normal hepatocytes. MMP had been restored by pretreatment of the hepatocytes with anti oxidants, showing the impact of reactive oxygen species in sumatriptan-induced mitochondrial damage ( Table 2). As expected, l-carnitine, TFP, l-Gln, fructose, cytochrome P450 enzyme inhibitors and/or endocytosis inhibitors restored MMP too.
Sumatriptan-induced mitochondrial damage was in turn increased by depleting hepatocyte GSH with 1bromoalkane, demonstrating the impact of GSH in sumatriptan induced mitochondrial damage.

Lysosomal Damage
A significant increase in lysosomal membrane damage was observed in hepatocytes after sumatriptan exposure which is associated with the leakiness of the lysosomal enzymes. This toxicity marker was significantly (p-value <0.05) inhibited by cytochrome P450 enzyme inhibitors, antioxidants, l-carnitine, TFP, l-Gln and fructose (Table  3). Lysosomal damage was in turn increased by depleting hepatocyte GSH with 1-bromoalkane, demonstrating the impact of glutathione in sumatriptan induced lysosomal damage. Significantly lower than sumatriptan-treated group (p < 0.05). Scale: 663 × 651.

Lipid Peroxides Production
Lipid peroxide induction which determined by measuring thiobarbituric acid reactive substances (TBAR) was significantly (p-value <0.05) increased after administration of sumatriptan. Lipid peroxidation has been significantly reduced by taurine, NAC and/or quercetin. Also, TBARS generation was significantly (p-value <0.05) prevented by the anti oxidants, endocytosis inhibitors, l-carnitine, TFP, l-Gln, fructose and/or cytochrome P450 enzyme inhibitors. Depletion of hepatocytes GSH with 1-bromoalkane in turn increased the TBARS levels, demonstrating the impact of glutathione in lipid peroxide production by sumatriptan in parenchymal hepatocytes (Table 4).

Reduced Glutathione Levels
180 min incubation of the cells with sumatriptan (5 mM) caused significant GSH depletion. As expected, GSH depletion has been significantly (p<0.05) restored with cytochrome P450 enzyme inhibitors, antioxidants, l-carnitine, TFP, l-Gln, fructose as well as lysosomal improver agents (Table 5).  Sumatriptan (5 mM  Several organs such as liver are involved in triptans adverse effects. [15][16][17] It is well known that imbalance between antioxidant defense and ROS generation (e.g., glutathione) to removal of ROS lead to oxidative stress. 49 GSH is an important antioxidant defense molecule for removal of reactive oxygen species such as lipid hydroperoxides and H 2 O 2 . 50 It has been demonstrated that sumatriptan affects superoxide release. 51 Surprisingly an in vitro study suggested that sumatriptan has scavenging activity on free radicals. 19 Also, it has been shown that the scavenging property is dosedependent. 52 Moreover, this medication has several metabolites after enzymatic processes such as indole acetic acid. Indole acetic acid derivatives are formed by sumatriptan metabolism in the liver 53 and one study demonstrated toxic effects of prooxidant radicals of indole acetic acid derivatives. 54 On the base of our results sumatriptan induces ROS formation and consequently depletes GSH. Our result also showed that GSH depletion by nontoxic bromoheptane via transferring the heptyl group of potent nontoxic bromoheptane to GSH form heptyl-Sglutathione and 39 caused a significant rise in sumatriptaninduced mitochondrial and lysosomal injury and consequently induced cell death. Migraine induces dysfunctional oxidative phosphorylation and consequently increases ROS formation. Several studies have shown the impact of antioxidants such as vitamin E in migraine. 55 Migraine pathogenesis is characterized by an increase in ROS generation. Thus migraine-induced ROS generation was significantly augmented after consumption of sumatriptan. Mitochondria have a key role in migraine. On the base of our results, sumatriptan toxicity is associated with GSH depletion and ROS generation and the antioxidants effectively reduced these toxicity markers. Mitochondrial dysfunction occurred during migraine. [56][57][58] It has been showed that mitochondrial ATP depletion results in MMP drop. 59 Normal MPT pore may be affected and opened by ROS formation. 60 While MPT is opened, some molecules such as cytochrome C that heretofore could not have crossed the mitochondrial membrane, now can release into cytosol and accelerate apoptosis. 61 Surprisingly, our results showed that MMP drop (% DΨm ) ensued after exposure to sumatriptan. This circumstance was prevented by lysosome improver, cytochrome P450 inhibitors, l-Gln, fructose, l-carnitine, TFP. Mitochondrial injury was significantly decreased by glutathione depletion and the MMP drop prevented by anti oxidants, suggesting that the MMP rapidly decreases after GSH depletion which in turn followed ROS formation. Table 3. Lysosomal membrane damage induced by sumatriptan in rat liver hepatocytes and protective effect of antioxidants, radical scavengers, lysosomal membrane stabilizers, mitochondrial ATP generators, MPT pore sealing agents and CYP450 inhibitors Lysosomal membrane fragility was measured as fluorescent intensity unit of diffused cytosolic green fluorescence induced by acridine orange following the redistribution from lysosomes into cytosol in acridine orange loaded hepatocytes. Results are expressed as mean ±S.E. of three separate experiments (n=3). a Significantly higher than control (p < 0.05). b Significantly lower than sumatriptan treated hepatocytes (p < 0.05). c Significantly higher than sumatriptan treated hepatocytes (p < 0.05).
On the base of their basic properties, chemicals such as drugs containing amine groups can be trapped into lysosomes. 62 As a result of this trapping, lysosomal membrane goes instable and consequently lytic enzymes such as proteases release into cytosol. Osmotic injury followed by membrane lysis is a common consequence of releasing of lysosomal content into cytosol. 38 Our results showed that hepatocytes incubation with sumatriptan leads to lysosomal injury that could be a result of accumulation of this chemical in lysosomes. 3-methyladenine and chloroquine are inhibitors of hepatocyte autophagy and used as lysosomal improver agents. 63 Our results showed that in addition to autophagy inhibitors, ROS scavengers and antioxidants significantly reduced lysosomal injury induced by sumatriptan in hepatocytes, suggesting that sumatriptan induced lysosomal injury can take place not only by direct effects of trapped sumatriptan in lysosomes but also after ROS formation. This was confirmed by an increase in lysosomal injury after GSH depletion. To author's knowledge, this is the first work which clarifies the impact of sumatriptan in lysosomal injury.

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Sumatriptan toxicity mechanisms Advanced Pharmaceutical Bulletin, 2016, 6(4), 627-637 Table 4. Lipid peroxidation induced by sumatriptan in rat liver hepatocytes and protective effect of antioxidants, radical scavengers, lysosomal membrane stabilizers, mitochondrial ATP generators, mitochondrial permeability transition pore sealing compounds and CYP450 inhibitors   Results are expressed as the means ± S.E of three separate experiments. a Significantly lower than control (p < 0.05). b Significantly higher than sumatriptan treated hepatocytes (p < 0.05).
CYP isoenzymes are important factors in ROS formation and can be involved in chemicals toxicity. 64 Triptans have several metabolites produced by liver enzymes. 65 It has been showed that sumatriptan pharmacokinetics is affected by CYP 3A4 inhibitors. 9 Also, quercetin was shown to inhibit the metabolism of chemicals by CYP3A4 in the liver. 66 Our results confirmed that pretreatment by CYP inducers phenobarbital caused a significant cell lysis which in turn prevented by cytochrome inhibitors 4-MP and/or cimetidine suggesting that CYP isoenzymes have an important effect in toxicity induced by sumatriptan and this should be considered in coadministration of sumatriptan and medications which induce or inhibit CYP enzymes.

Conclusion
In conclusion our results suggest that anti oxidants and ATP generators seems to be useful medicines for improving triptan efficacy and reducing toxicity induced by these drugs and it is proposed that prescription of appropriate anti oxidants and ATP generators can be included in migraine therapy. Additionally since taurine and N-acetylcysteine are available in drugstores from various pharmaceutical companies, simultaneous prescription of this supplements with sumatriptan is possible. Moreover, it is suggested that the impact of this medication on cell organelles should be studied by details in animal models of migraine headache and aura regarding the oxidative stress induced by migraine.