CISPLATIN IS ONE OF the most widely used and most effective cytotoxic agents in the treatment of a variety of malignancies, including lung, colorectal, ovarian, breast, head/neck, bladder, and testicular cancers in both children and adults.[1, 2] Similarly to other anti-cancer therapeutics, cisplatin treatment is associated with significant toxicities consisting of myelotoxicity, nephrotoxicity, neurotoxicity, and ototoxicity. Nephro- and neurotoxicity, however, are the major side-effects of cisplatin treatment that hinder its use in the clinic. Neurotoxicity occurs in a dose-dependent manner, is cumulative, and arises in approximately 50% of patients receiving cisplatin.[4, 5] Cisplatin-mediated neurotoxicity is therefore a significant limitation to effective anti-cancer treatment because it may lead to dose reduction or even treatment withdrawal. Therefore, effective strategies to reduce the severity of nerve damage following chemotherapy are intensively being searched for.
The chemotherapeutic mechanism of cisplatin is through DNA binding, inducing DNA damage that leads to apoptosis. The classical target of cisplatin neurotoxicity has also been well established (i.e. dorsal root ganglia neurons). In addition, excitotoxic glutamate release, neuronal expression of copper transporter 1, interference with normal cell metabolism, formation of platinum-DNA-protein cross-links, polymorphisms in the DNA repair genes and, finally, activation of the Rho GTPases have all been found to be associated with cisplatin-induced neurotoxicity.[2, 8-12] Another possible, although less well-documented, common site of action of this compound is represented by mitochondria. Lately, evidence that mitochondrial dysfunction can be induced, probably through binding to mitochondrial DNA by cisplatin, has been reported. Mitochondrial dysfunction here increases the oxidative stress, and oxidative stress plays an important role in the pathogenesis of cisplatin neurotoxicity.[14, 15] Moreover, several antioxidant treatments have been used successfully in order to prevent cisplatin-mediated neurotoxicity.[16-18] Some protective agents, however, may interfere with the anti-tumoral effects of cisplatin. New treatment strategies that do not interfere with the anti-neoplastic effects of cisplatin to limit cisplatin neurotoxicity are therefore always necessary.
In contrast, mirtazapine, as a noradrenergic and specific serotonergic antidepressant, may be a promising agent for the avoidance of cisplatin-induced neurotoxicity. Mirtazapine is used to treat major depressive disorder effectively in clinical psychiatric practice. Mirtazapine also has a less-known additional feature of repression of the production of enzymatic and non-enzymatic oxidation indicators while increasing the antioxidant parameters. Mirtazapine has recently been reported to protect gastric tissue from the gastrotoxic effect of indomethacin via its antioxidant properties. Furthermore, we recently found that mirtazapine ameliorated cisplatin-induced nephrotoxicity in rats. These findings suggest that mirtazapine could be a cytoprotective agent, but no systematic data on the defensive effect of mirtazapine with regard to the cisplatin neurotoxicity have been reported to date. In the present study, we examined the potential chemoprotective effects of mirtazapine against cisplatin-induced oxidative stress and DNA damage in rat brains by biochemical methods.
Whole biochemical assay compounds were purchased from Sigma (Munich, Germany) and Merck (Darmstadt, Germany). Additionally, mirtazapine was bought from Merck Sharp and Dohme (Istanbul, Turkey), and cisplatin was obtained from Orna (Istanbul, Turkey).
All experiments were performed with 24 adult male Wistar Albino rats, each weighing 210–230 g. They were obtained from Ataturk University Experimental Animal Laboratory of Medicinal and Experimental Application and Research Centre. Care of the rats was in accordance with the ILAR/NRC Guide for the Care and Use of Laboratory Animals (2010 edition), and all procedures were also approved by the Ataturk University Local Animal Care Committee. The Ethics Committee of the Ataturk University Medical Faculty approved the whole study protocol. Rats were housed in standard plastic cages on sawdust bedding in an air-conditioned room at 22 ± 1°C under controlled lighting (14 h light/10 h dark cycle). Standard rat chow and tap water were given ad libitum. The rats were divided equally into four groups: control, cisplatin, cisplatin plus mirtazapine, and mirtazapine. The groups were separated in different cages.
The cisplatin plus mirtazapine group and the mirtazapine group were first given 30 mg/kg mirtazapine in distilled water by gastric gavage while the animals in the control and the cisplatin groups received solely distilled water in equal volume by the same route. One hour after this procedure, 10 mg/kg cisplatin, which was dissolved in isotonic saline solution, was injected i.p. every day for 14 days into the animals in the cisplatin and the cisplatin plus mirtazapine groups, while the animals in the control and the mirtazapine groups were given only isotonic saline solution in equal volume on the same schedule. All animals were killed by high-dose anesthesia (50 mg/kg thiopental sodium) at the end of the 14 days; their brains were removed quickly and analyzed for biochemistry as soon as possible.
The present study was carried out with a high dose of cisplatin (10 mg/kg body weight, i.p.) corresponding to the dose of cisplatin currently being used in clinical practice. The corresponding dose in human being (60 kg) is 30 mg/m2. The onset of toxicity, however, is delayed in humans until a cumulative dose >300 mg/m2 has been given. Therefore, we extended the treatment duration to guarantee induction of oxidative stress and, consequently, neurotoxicity. We also worked under exactly the same conditions (i.e. chemicals, animals, pharmacological procedures, biochemical procedures, laboratories, and staff) as in our previous nephrotoxicity study.
The amount of glutathione (tGSH) in the total homogenate was measured according to the method of Sedlak and Lindsay with some modifications.
Nitric oxide end products
NO end product levels were measured using the Griess reaction[25, 26] with further biochemical techniques. The details of the procedure were described in one of our recent studies.
The concentrations of lipid peroxidation were determined by estimating malondialdehyde (MDA) using the thiobarbituric acid test.
Myeloperoxidase (MPO) activity was measured according to the method of Bradley et al.
Brain tissue was drawn and DNA isolated using the modified method of Shigenaga et al. Approximately 50 mg of DNA was hydrolyzed with 0.5 mL of formic acid (60%, v/v) for 45 min at 150°C. The amount of 8-hydroxyguanine (8-OH-GUA) and GUA was measured using a high-performance liquid chromatography system equipped with an electrochemical detector (HP Agilent 1100 module series, ECD HP 1049A), as described previously.[31, 32] GUA and 8-OH-GUA (25 pmol) were used as standards. 8-OH-GUA level is expressed as the number of 8-OH-GUA molecules/105 GUA molecules. 8-OH-GUA is a marker of oxidative DNA damage here.
Statistical evaluation was performed using SPSS (Chicago, IL, USA) v18.0 for Windows. The data were analyzed using analysis of variance (ANOVA) followed by post-hoc Bonferroni tests when comparing the four groups. Statistical significance was set at P < 0.01.
On ANOVA it was found that levels of tGSH, NO end products, MDA, MPO, and 8-OH-GUA significantly differed across groups (Table 1). Findings obtained with the Bonferroni multiple comparison tests were as follows. tGSH was lower in the cisplatin group than the control group, the cisplatin plus mirtazapine group, and the mirtazapine group (72.44%, 85.90%, and 90.06% percentage changes [PC], respectively). Similarly, the level of NO end products were higher in the control group, the cisplatin plus mirtazapine group, and the mirtazapine group (61.99%, 55.30%, and 62.77% PC, respectively). MDA level was higher in the cisplatin group than the control group, the cisplatin plus mirtazapine group, and the mirtazapine group (−55.48%, −60.50%, and −56.37% PC, respectively). MPO level was higher in the cisplatin group than the control group, the cisplatin plus mirtazapine group, and the mirtazapine group (−67.99%, −78.59%, and −79.69% PC, respectively). In order from high to low MPO level, the groups were: cisplatin, control group, and cisplatin plus mirtazapine. MPO level did not differ within the cisplatin plus mirtazapine group and the mirtazapine-alone group. Finally, 8-OH-GUA level was higher in the cisplatin group than the control group, the cisplatin plus mirtazapine group, and the mirtazapine group (−48.81%, −38.10%, and −49.40% PC, respectively). The level of 8-OH-GUA in both the control and mirtazapine groups was lower than in the cisplatin group and the cisplatin plus mirtazapine group. The combination cisplatin–mirtazapine group had significantly lower 8-OH-GUA than the cisplatin group.
Table 1. Biochemical parameters (one-way ANOVA) vs group
|tGSH||5.38||0.28||3.12||0.34||5.80||0.61||5.93||1.15||57.928||0.000||A = C = D > B|
|NO end products||10.40||0.49||6.42||0.19||9.97||0.56||10.45||0.42||117.709||0.000||A = C = D > B|
|MDA||2.48||0.26||5.57||0.31||2.20||0.29||2.43||0.22||208.789||0.000||B > A = C = D|
|MPO||1.45||0.19||4.53||0.33||0.97||0.21||0.92||0.17||334.849||0.000||B > A > C = D|
|8-OH-GUA||0.86||0.07||1.68||0.06||1.04||0.04||0.85||0.07||242.756||0.000||B > C > A = D|
The continued clinical use of cisplatin is limited by the onset of severe neurotoxicity. Cisplatin-induced neurotoxicity is associated with histological damages and behavioral alterations.[35-38] Neurotoxicity has been observed in approximately 50% of patients treated with cisplatin. A great effort has been made to resolve this issue with regard to optimal dose and duration, especially in recent years. For this purpose, various methods have been investigated to prevent the neurotoxic side-effects of cisplatin. Among the compounds with potential neurotrophic action, several antioxidants have shown a great range of neuroprotective effects both in vitro and in vivo.[16-18] Although the exact mechanism of cisplatin-induced neurotoxicity is still not completely understood, however, there is general agreement that it depends on the induction of apoptosis in neuronal cells. Presumably, irreversible cell damage and apoptosis might result mainly from oxidative stress. Therefore, trials of antioxidant supplements in cisplatin-induced neurotoxicity have been carried out.
The previous success of antioxidants in this area, however, also supports a central role of oxidative stress in the pathogenesis of cisplatin-mediated neurotoxicity indirectly. In line with this inference, we found that although the tGSH and NO end product mean scores were significantly decreased, MDA and MPO mean scores were statistically higher in the cisplatin group when compared with the control group. tGSH here plays an important role in a number of critical cellular processes, including providing the major line of defense for the protection of cells from oxidative and other forms of stress. tGSH can scavenge free radicals, reduce peroxides and be conjugated with electrophilic compounds. It thereby provides cells with multiple defenses against both reactive oxygen species (ROS) and their toxic by-products. It has also been demonstrated that cisplatin induces cell death, which is characterized by reduced tGSH level, which is suggestive of accelerated oxidative processes not only in the brain, but also in other tissues previously.[15, 21, 42-45] When cisplatin enters cells, adduct formation between cisplatin and tGSH can occur, and tGSH may decrease as a consequence.
In the last decade, among signal molecules interest has grown in NO in the central nervous system (CNS) due to NO involvement in the maintenance and regulation of normal neuronal processes, including those related to the toxic conditions. NO has also been suggested to play an important role in cisplatin-induced cytotoxicity. Similarly, cisplatin has been shown to cause a decrease of NO activity in various tissues, as well as in the brain.[47-49] In the mammalian CNS, the NO-producing enzyme, that is, neuronal nitric oxide synthase (nNOS), is Ca2+/calmodulin dependent. Previously, a direct interaction between hydrolyzed forms of cisplatin and the calcium-binding sites of the calmodulin molecule in the stomach of the rat has been demonstrated: this interaction, indirectly, inhibited nNOS activity in the pyloric sphincter. We may deduce that a similar mechanism may also compromise the nNOS activity of the brain in cisplatin-treated rats, resulting in a decrease in the level of NO end products. In line with this inference, Kim et al. showed that the expression of nNOS decreased in the renal inner medulla following treatment of cisplatin. The selective downregulation of nNOS here may play a role in decreased medullary blood flow and secondary tissue injury. In addition, l-arginine, the precursor of NO in the body, attenuates cisplatin-induced nephrotoxicity, and a competitive inhibitor of NOS, NG-nitro-l-arginine methyl ester hydrochloride (l-NAME), exacerbates cisplatin-induced nephrotoxicity.[52, 53] In contrast, l-NAME and 7-nitroindazole, a nNOS inhibitor, have also been shown to significantly suppress oxaliplatin-induced pain behavior in rats. But another possible, although probably less pronounced, explanation is that the expression and/or activity of brain inducible nitric oxide synthase (iNOS) compared with kidney iNOS did not reach the expected level even under these oxidative stress conditions induced by cisplatin treatment. Therefore, both beneficial and toxic effects of NO have been suggested in cisplatin-induced cytotoxicities, and the exact role of NO in these experimental models remains controversial. Further studies are needed in order to confirm the present findings.
MDA is one of the end products of lipid peroxidation and an indicator of ROS production. Therefore, an increase in MDA tissue level indicates an increase in free oxygen radicals. The most important and damaging cellular effect of free radicals is lipid peroxidation. MDA in its turn causes more advanced cellular injury. Cisplatin treatment can cause marked organ damage, characterized by a significant increase in tissue MDA level compared to normal control.[21, 56-60] In summary, elevation of MDA in different tissues seems to be a result of the direct peroxidative effect of cisplatin use.
Similarly, cisplatin injections caused significant elevation in tissue MPO activities compared to control values.[21, 61-63] MPO is found in phagocytic cells. As an enzyme, MPO catalyses the production of the highly chlorinating and oxidizing agent hypochlorous acid from chloride and peroxide, which reacts with the low-density lipoprotein apoprotein moiety, leading to the derivatization of its aminoacid residues. Therefore, excess production of MPO and other reactive radicals causes oxidative damage. Additionally, it is also used as an indication of tissue neutrophil accumulation. In all these contexts, alteration of MPO activity might be associated with increased neurotoxicity.
To our knowledge, we have shown that brain tissue 8-OH-GUA level was increased in the rat cisplatin group in comparison to the rat control group for the first time in the literature. ROS are known to induce several types of oxidative DNA damage, including 8-OH-GUA. Notably, aforementioned recent studies indicated that cisplatin acted as a pro-oxidant. Therefore, we can speculate that 8-OH-GUA accumulation in the brain tissue reflects an imbalance between DNA damage and repair. In short, the disturbance of DNA repair enhances the accumulation of 8-OH-GUA. The overproduction of 8-OH-GUA is also thought to be responsible for apoptosis. In summary, cisplatin exerts its neurotoxic effects via oxidative stress predominantly. All of the previous antioxidant therapies, however, have not been able to achieve good results in the fight against cisplatin neurotoxicity.
Three main features of the possible defending antioxidant agents came into prominence during these studies. First, because the anti-tumoral properties of cisplatin originate mainly from its binding to DNA, which causes significant distortion of the helical structure and results in the inhibition of DNA replication and transcription, some of the protective molecules that disrupt this special relationship might also reduce the anticancer qualities of cisplatin. For instance, Fischer et al. showed that cisplatin binds rapidly to dimethyl sulfoxide (DMSO) to form a DMSO adduct. The resulting compound has a reduced ability to bind to double-stranded DNA both in vitro and in cells. This compound has reduced toxicity for both cancer cells and neurons in vitro. Additionally, cisplatin also induces apoptosis via activation of caspase 3.[69-72] Caspase 3 can be activated by caspase 9, which is activated by the release of cytochrome c from the mitochondria.[73, 74] Thiol-based antioxidants such as sodium thiosulfate block these apoptotic pathways induced by cisplatin and decrease anti-tumor efficacy while protecting against neurotoxicity. Second, although the platinum drugs have poor penetration through the blood–brain barrier, levels of cisplatin have been shown to be sufficient to cause toxicity in the brain. For example, although CNS toxicity associated with cisplatin chemotherapy is characterized by acute encephalopathy, stroke-like episodes, acute blindness, and seizures in humans, it has been shown that use of systemic cisplatin in mice is associated with increased cell death and decreased cell division in the subventricular zone, in the dentate gyrus of the hippocampus, and in the corpus callosum of the CNS.[2, 76, 77] For this reason, the antioxidant compounds should cross blood–brain barrier so that the drug can be given peripherally in the treatment of neurotoxicity. Third, of course, protective antioxidant molecules are expected not to be toxic. For instance, Church et al. found that although high doses of amifostine provided the beneficial effect of protecting against cisplatin-induced ototoxicity, it had the harmful side-effect of neurotoxicity. Thus, new treatment alternatives that do not interfere with the anti-neoplastic effects of cisplatin; are not stopped by the blood–brain barrier; and have not their own toxic side-effects to limit cisplatin-mediated neurotoxicity are always needed.
For this purpose, we have preferred to use mirtazapine for its possible protective effects against oxidant stress due to cisplatin treatment in the present study. The results showed that brain antioxidant defense systems, such as tGSH and NO end products that are depleted by cisplatin therapy, were restored to normal by treatment with mirtazapine. Cisplatin-induced lipid peroxidation, that is, MDA level, was also found to be markedly reduced by treatment with mirtazapine. Furthermore, mirtazapine decreased leukocyte/macrophage infiltration in rat brain tissue considerably (i.e. activity of MPO). And finally, mirtazapine ameliorated DNA damage (significant decline in 8-OH-GUA level), indicating that damage was probably mediated by the oxidative stress. Bilici et al. recently investigated the anti-ulcer effects of mirtazapine and determined its relationship with antioxidant mechanisms. In agreement with the present findings, they found that although mirtazapine treatment significantly increased tGSH in the stomach tissue compared to the control, the gastric MDA and MPO levels significantly decreased with mirtazapine therapy in comparison with distilled water. We investigated a possible role of oxidative stress in the pathogenesis of cisplatin-induced nephrotoxicity and tested the potential protective effects of mirtazapine against this harmful side-effect in rat kidneys by biochemical and histological methods. While tGSH level was restored to normal, MDA and MPO levels were again significantly reduced by treatment with mirtazapine. In this context, activation of enzymatic and non-enzymatic antioxidant mechanisms and inhibition of some toxic oxidant mechanisms might also have a role in the defense of mirtazapine against cisplatin neurotoxicity. The latter fact, however, does not mean that the neuroprotective effect of mirtazapine is due to a single mechanism of action. Mirtazapine also blocks 5-hydroxytriptamine2/3 (5-HT2/3) receptors specifically and blockade of these receptors may contribute to the protective properties of mirtazapine, because some of the substance-induced neurotoxicities are mediated via 5-HTergic transmission involving 5-HT2/3 receptors.[56, 79-81] Interestingly, the MPO mean score of the cisplatin plus mirtazapine group was statistically lower than the control group. The MPO-reducing effect of mirtazapine might be explained by its potent antioxidant properties, as shown in previous studies.[20, 82] We also found a significant difference between the control group and the cisplatin plus mirtazapine group for 8-OH-GUA. Therefore, the 8-OH-GUA mean score of the control group was higher than the cisplatin plus mirtazapine group. Surely, mirtazapine could not prevent the entire DNA damage via its antioxidant and 5-HT2/3 receptor antagonistic qualities, because the anti-neoplastic mechanism of cisplatin is through DNA binding, inducing DNA damage, and mirtazapine does not have a special effect on this relationship. Also, mirtazapine also reduces nausea, anorexia, sleep disturbance, as well as depression in cancer patients.[83-86] All these positive effects might provide a new indication for mirtazapine, and it might become one of the first-choice drugs in depressive patients receiving cisplatin chemotherapy.
In summary, the present results suggest that although oxidative stress plays a central role in the pathogenesis of cisplatin-mediated neurotoxicity, mirtazapine has protective effects against this severe situation, which may be attributed to its antioxidant and 5-HT2/3 receptor antagonistic potentials. Mirtazapine might also be a cytoprotective agent and therefore it would be useful to investigate whether cisplatin at the desired doses can be given concurrently with mirtazapine in the absence of other considerations. We were unable, however, to compare the present findings with others because no other experimental study in the literature has yet investigated the protective effects of mirtazapine on cisplatin-induced neurotoxicity in rat brain. Demonstration of the possible histological and/or behavioral benefits of reducing oxidative stress with mirtazapine is advisable in the future. In contrast, large-scale prospective studies that examine the association between cisplatin neurotoxicity and the protective effects of mirtazapine are also needed in order to confirm the present findings in humans.