http://annalsofrscb.ro 17537
Assessment the oxidative stress potential of fluoxetine and amitriptyline at maximum therapeutic doses for four-week
treatment in experimental male rats
Imad Adnan Abd Al-Obaidi *, Noor A. Mohammed Hassan ¹, and Prof. Dr. Nada N.
Al-Shawi 2
* Medico-Legal Directorate, Baghdad, Iraq.
1 Medical City Directorate, Baghdad, Iraq.
2 Department of Pharmacology and Toxicology, College of Pharmacy, University of Baghdad, Baghdad, Iraq.
* Corresponding author E-mail: [email protected] Abstract
Nowadays, the contribution of oxidative stress (OS) to the safety profile of medications at a specific doses remains widely undetermined. Antidepressants are known to be used over a long- term periods and the incidence of their toxicities due to accumulative OS cannot be excluded.
Therefore, the aim of this study was to investigate the possible oxidative damage of the commonly used antidepressants (fluoxetine and amitriptyline) besides their histopathological effects in adult male rats.
Selected parameters of OS (MDA and SOD) in addition to histopathological changes have been examined in liver and testis tissues of 24 Swiss albino adult male rats; the animals were randomly allocated into three groups of 8 rats each: Group I - rats orally-administered distilled water via gavage tube for four weeks as a negative control. Group II - rats orally-treated with fluoxetine hydrochloride solution (7.2mg/kg/day) via gavage tube for four weeks. Group III - rats orally- treated with amitriptyline hydrochloride solution (27mg/kg/day) via gavage tube for four weeks.
The results revealed that both drugs (Group II and Group III) induced the same extent of oxidative damage, as evidenced by a significant elevation of MDA contents and a marked reduction of SOD levels in hepatic and testicular tissue homogenates as compared with the negative control (Group I), which have been confirmed by histopatholgical alterations.
These findings indicates that both fluoxetine and amitriptyline have cytotoxic potentials and can induce the same extent of oxidative damage in rats. Special precautions and medical supervision should be taken in consideration with their uses.
Keywords: Oxidative stress, fluoxetine, amitriptyline, MDA, SOD, Histopathology.
Introduction
Depression and anxiety disorders are serious public health illnesses (Tiller, 2013), with approximately 350 million people affected around the world (Pilania et al., 2013). As mentioned by the World Health Organization (WHO), the burden of depression will rise globally and
http://annalsofrscb.ro 17538 predicted to be the second largest burden after ischemic heart disease by 2020 (Gupta, Nihalani and Masand, 2007). At that point, it is time to pause and reflect because with more people suffering from anxiety and depression, the use of antidepressant medications will be rise widely, and their consumption will growing worldwide (Ali and Hendawy, 2018). Substantial multinational analyses of routinely prescribed antidepressants have found that fluoxetine and amitriptyline are two of the most widely prescribed antidepressants belonging to selective serotonin reuptake inhibitors (SSRI) and tricyclic antidepressants (TCAs) groups, respectively (Chee et al., 2015)(Tamblyn et al., 2019).
Unfortunately, there have been several studies showing collateral health effects caused by fluoxetine and amitriptyline uses (Rajamani et al., 2006)(Deidda et al., 2017)(Jilani et al., 2013)(Sorodoc et al., 2013)(Moreno-Fernández et al., 2008), among these undesirable effects are hepatic and reproductive disorders (Inkielewicz-Stępniak, 2011)(Bataineh and Daradka, 2007)(Afify et al., 2010).
Accordingly, in 2004 expert panel report on the reproductive and developmental toxicity of fluoxetine from the NTP Center for the Evaluation of Risks to Human Reproduction (CERHR) revealed that sufficient evidence exists to conclude that fluoxetine can produce developmental toxicity and reproductive toxicity in males and females (Hines et al., 2004). Similarly, in the same year the American Association of Poison Control Centers reported 12,234 cases of TCA toxicity and 61% of them was toxicity of amitriptyline (Basol and Erbas, 2016).
Since the antidepressants are medications which can be consumed regularly for 6 months or more, with a potential recurrence of the treatment (Bet et al., 2013). Therefore, it is of great importance to carry out this study on commonly used antidepressants (fluoxetine and amitriptyline) to assess their OS impact.
Materials and Methods
Chemicals and Drugs
Fluoxetine and amitriptyline as hydrochloride powders were purchased from Sigma-Aldrich Co., St. Louis, MO, USA. All other chemicals used were of analytical grade.
Preparations of Drugs Treatment Solutions
Fluoxetine and amitriptyline hydrochloride solutions were freshly-prepared every day by dissolving the required amount of each of drug powder in sterile distilled water to get a final concentration (7.2 mg/kg and 27 mg/kg B.wt per day) of fluoxetine and amitriptyline, respectively. The doses of fluoxetine and amitriptyline were calculated by extrapolating the human recommended maximum therapeutic doses to rat doses, according to the conversion table of Paget and Barnes (Paget and Barnes, 1964).
Experimental animals
http://annalsofrscb.ro 17539 The study was performed on 24 healthy experimental Swiss Albino adult male rats, weighing (200-300 g), in accordance with the guidelines of the Biochemical and Research Ethical Committee; and approved by the Scientific Committee at the Department of Pharmacology and Toxicology, College of Pharmacy, University of Baghdad. The animals were supplied by the Animal House of the College of Pharmacy, University of Baghdad. All animals were housed within plastic cages and maintained under standard laboratory conditions at temperature 22-24°C under a 12-h light/dark cycle, and offered a free access to food (commercial rat pellets) and water ad libitum. After 3 days of acclimation, experimental rats were randomly allocated into three groups of 8 rats each, as follows: Group 1: Rats orally-administered distilled water (DW) daily via gavage tube for four weeks. This group served as negative control. Group 2: Rats orally- administered a maximum therapeutic dose of fluoxetine hydrochloride solution (7.2mg/kg/day) via gavage tube for four weeks. Group3: Rats orally-administered a maximum therapeutic dose of amitriptyline hydrochloride solution (27mg/kg/day) via gavage tube for four weeks. After 24 h of the end of the treatment duration (i.e. at day 29), rats were euthanized by diethyl ether and sacrificed by cervical dislocation. Livers and testes were excised, weighed and washed with normal saline 0.9%. A small piece of liver about 2 gram and the left testis were preserved in chilled phosphate buffer saline (1X PBS) and kept frozen until further analysis. A small piece of liver about 2 gram and the right testis were fixed in 10% neutral buffered formalin for histological examinations.
In Vivo Oxidative Stress Study Measurement of lipid peroxidation
Measuring the end product of lipid peroxidation is one of the most widely accepted assays to assess OS in pathophysiological processes. The Quantification of lipid peroxidation was estimated in liver and testis by measuring MDA content using a commercial MDA Microplate Assay Kit (Cohesion Biosciences, UK, 542-78-9), according to the method described by Ohkawa H, Ohishi N and Yagi K (1979) (Ohkawa, Ohishi and Yagi, 1979). The MDA in the sample is reacted with thiobarbituric Acid (TBA) to generate the MDA-TBA adduct, which can easily be quantified colorimetrically at (λ = 532 nm).
Determination of superoxide dismutase (SOD) activity
Superoxide dismutase (SOD) activity in the liver and testis was determined using a commercial Superoxide Dismutase Microplate Assay Kit (Cohesion Biosciences, UK, 9054-89-1), according to the method described by Charles Beauchamp and Irwin Fridovich (1971) (Beauchamp and Fridovich, 1971). Superoxide Dismutases (SOD) catalyze the dismutation of the superoxide radical (O2
.-) into hydrogen peroxide (H2O2) and elemental oxygen (O2). The superoxide radical (O2
.-) generated from the conversion of xanthine to uric acid, and hydrogen peroxide by xanthine oxidase (XO), convert nitroblue tetrazolium (NBT) to NBT-diformazan. NBT-diformazan absorbs light at 560 nm. SODs reduce superoxide ion concentrations and thereby lower the rate of
http://annalsofrscb.ro 17540 NBT-diformazan formation. The extent of reduction in the appearance of NBT-diformazan is a measure of SOD activity present in experimental samples.
Histological examination
Sections of liver and testis were prepared for histological examination according to the method described by Bancroft and Gamble (2008) (Bancroft and Gamble, 2008) using paraffin sections technique.
Statistical Analysis
Statistical analysis of data was performed using SAS (Statistical Analysis System-version 9.1).
Descriptive statistics for the numerical data were expressed as mean and standard deviation (mean ±SD). One way and two ways Analysis of Variance (ANOVA) and Least significant difference post-hoc test were used to assess the significant differences among groups. P< 0.05 is considered statistically significant (‘SAS/STAT Users Guide for Personal Computer. Release 9.13’, 2010).
Results
Effects on selected oxidative stress parameters (MDA and SOD)
Analyzing the data by two-way ANOVA test revealed that contents of MDA in liver and testes were significantly elevated (P<0.05) in rats treated with fluoxetine (Group 2) and amitriptyline (Group 3) each compared to the negative control (Group 1) rats. In addition, there were non- significant differences (P>0.05) in contents of MDA between liver and testes tissues in male rats treated with fluoxetine; while, there was a significant increment (P<0.05) in hepatic MDA contents compared to the testicular MDA contents of rats in amitriptyline-treated group (Table 1).
Table 1. Contents of malondialdehyde (MDA) in liver and testes tissues of male rats.
Groups (N=8)
MDA nmol/g Tissue liver
MDA nmol/g Tissue testes
Control 6.64±0.84 b A 6.13±0.42 b A
Fluoxetine 12.09±1.14 a A 12.13±1.82 a A
Amitriptyline 15.20±1.90 a A 12.82±1.32 a B
Data are expressed as (mean ± SD); n=8 animals in each group;
Means with a different small letters superscripts (a, b) in the same column are significantly different (P<0.05);
Means with a different capital letters superscripts (A, B) in the same row are significantly different (P<0.05).
http://annalsofrscb.ro 17541 A two-way ANOVA analysis revealed that SOD levels in liver and testes were significantly reduced (P<0.05) in animals treated with fluoxetine and amitriptyline each compared with the control animals. In addition, there were non-significant differences (P>0.05) in hepatic SOD levels compared to the testicular SOD levels of rats in fluoxetine- and amitriptyline-treated groups. (Table 2).
Table 2. Levels of superoxide dismutase (SOD) enzyme activity in liver and testes tissues.
Groups SOD U/g Tissue liver
SOD U/g Tissue testes
Control 18.30±3.42 a A 12.42±1.87 a B
Fluoxetine 6.92±1.22 b A 5.68±1.02 b A
Amitriptyline 5.29±1.83 b A 4.56±1.16 b A
Data are expressed as (mean ± SD); n=8 animals in each group;
Means with a different small letters superscripts (a, b) in the same column are significantly different (P<0.05);
Means with a different capital letters superscripts (A, B) in the same row are significantly different (P<0.05).
Effects on Hepatic and Testicular Histology
Microscopic examination of liver section of (Group 1) male rats orally-received D.W showed a normal hepatic architecture with cords of hepatocytes surrounding the central vein and the hepatic sinusoids radiating in between the densely packed cells. Liver sections from male rats treated with fluoxetine (Group 2) showed several histological alterations, including apoptotic hepatocytes, dilated sinusoids, increased number of kupffer cells, slight hepatocyte vacuolation and mild central vein congestion. Liver sections from male rats treated with amitriptyline (Group 3) showed several histological changes, including apoptotic hepatocytes, dilated sinusoids, increased number of kupffer cells, slight hepatocyte vacuolation and inflammatory cells infiltration (Figure 1).
http://annalsofrscb.ro 17542 Figure 1. Light microscopic liver sections of (A) Control rats showing normal hepatic tissue; (B) Fluoxetine-treated rats showing apoptotic hepatocytes (blue arrow), hepatocyte vacuolation (yellow arrow), mild central vein congestion (red arrow) and dilated sinusoids with increased number of kupffer cells (white arrow); (C) Amitriptyline-treated rats showing apoptotic hepatocytes (blue arrow), hepatocyte vacuolation (yellow arrow), inflammatory cells infiltration (black arrow) and dilated sinusoids with increased number of kupffer cells; hepatocyte (H), central vein (CV), bile duct (BD), hepatic sinusoid (S), H&E (40X).
The microscopic examination of the negative control testicular sections (Group 1) male rats revealed normal spermatogenic activity that characterized by normal seminiferous tubules with densely-packed germinal epithelium and the lumen filled with sperms were indicated. The testicular tissues from fluoxetine-treated male rats (Group 2) showed disrupted seminiferous tubules with marked reduction in germinal epithelium, signs of cellular degeneration and seminiferous vacuolation. The testicular tissues from amitriptyline-treated male rats (Group 3) showed disrupted seminiferous tubules with marked reduction in germinal epithelium, signs of cellular degeneration, seminiferous vacuolation and decreased sperms number in some tubules (Figure 2).
http://annalsofrscb.ro 17543 Figure 2. Light microscopic testes sections of (A) Control rats showing normal testicular tissue;
(B) Fluoxetine-treated rats showing disrupted seminiferous tubules with marked reduction in germinal epithelium (green arrow) and seminiferous vacuolation (yellow arrow); (C) Amitriptyline-treated rats showing disrupted seminiferous tubules with marked reduction in germinal epithelium (green arrow), slight decrease in sperms number (red arrow) and seminiferous vacuolation (yellow arrow); germinal epithelium (GE), sperms inside lumen (S), H&E (40X).
Discussion
Effects of fluoxetine and amitriptyline on oxidative stress parameters
In the present study, fluoxetine caused a significant increase in MDA contents and significant decrease in SOD levels of hepatic and testicular tissues of the treated animals as shown in (Table 1 and Table 2).
In order to determine the most likely mechanism behind such impaired redox state, it seems that the mitochondrial dysfunction induced by fluoxetine treatment play a major role in hepatic oxidative damage, which confirmed clearly by the previous study of Souza et al (1994) (Souza et al., 1994).
It has been established that mitochondria are the main source of cellular ROS production in the hepatocytes (Cichoż-Lach and Michalak, 2014), and OS is always associated with mitochondrial dysfunction. The electron transport chain is blocked in the damaged mitochondria, resulting in accumulation of ROS, consumption of antioxidants, energy depletion, accumulation of cytotoxic mediators, creating a vicious circle and cell death (Lee, Giordano and Zhang, 2012)(Wang et al., 2016).
In testes, spermatozoa are the primary target cells for OS; and are extremely sensitive to ROS and constantly exposed to oxidizing environments due to their high content of polyunsaturated fatty acids (PUFAs) and their limited ability to restore the oxidative damage and hence, lipid peroxidation (Agarwal et al., 2014).
In support of these facts, the testicular OS induced by fluoxetine can be interpreted in view of the in vitro study of Kumar et al (2006) (Kumar et al., 2006), who mentioned that fluoxetine inhibit
http://annalsofrscb.ro 17544 oxidative phosphorylation in sperm mitochondria and interact indirectly with phospholipids in the inner mitochondrial membrane which may arise due to its possible ability to interact with the sulfhydryl groups present over sperm membrane.
While, Safarinejad (2008) (Safarinejad, 2008) reported that, such testicular oxidative damage induced by SSRIs may account for 5-HT capability to reduce cupric ions (Cu+2) with subsequent generation of ROS, such as the hydroxyl radical (·OH).
Concerning effects of amitriptyline, such drug shows a significant increase in MDA contents and a significant decrease in SOD levels in hepatic and testicular tissues of the treated animals as shown in (Table 1 and Table 2).
The hepatic findings of amitriptyline could be interpreted in view of the research of Bautista- Ferrufino et al (2011) (Bautista-Ferrufino et al., 2011), who found that amitriptyline-induced hepatic OS in mice was accompanied by significant elevation of MDA levels, as a consequence of coenzyme Q deficiency and mitochondrial dysfunction induced by amitriptyline treatment for two weeks.
Supporting the present findings, a study conducted by Bandegi et al (2018) (Bandegi et al., 2018) provide more assurance about amitriptyline-induced testicular OS in orally-treated mice. In such study, long term treatment with amitriptyline at dose of 4 mg/kg B.wt. produced a marked increase in MDA levels as an indicator of lipid peroxidation and a significant decline in the total antioxidant capacity levels.
Moreover, varying levels of MDA contents between liver and testis tissues that have been induced by amitriptyline in the present study, could be interpreted in view of the recent study by Kampa et al (2020) (Kampa et al., 2020), who found that oral administration of amitriptyline for 2 weeks in male rats caused hepatic lipids upregulation in the periportal area indicating that amitriptyline induced phospholipidosis and macrovesicular steatosis, besides its induced liver injury. As a result, these upregulated lipids of liver will become more prone to lipid peroxidation that has been indicated by significantly higher levels of MDA contents as compared to those of testis.
Effects of fluoxetine and amitriptyline on liver and testis histology
It has been well documented that DNA damage or mitochondrial damage are serious cellular stressors that can trigger apoptosis through activation of p53 protein and other transcriptional factors (Rastogi and Sinha, 2009). Thus, evidence of apoptotic hepatocytes in the present work confirms cytotoxic potential that have been induced by fluoxetine and amitriptyline.
Moreover, It has been emphasized that OS and inflammation are tightly correlated and orchestrated in the development of hepatic damage (Li et al., 2016), which clearly explains the increased number of kupffer cells and central vein congestion that have been demonstrated in the present study.
Kupffer cells, the hepatic macrophages considered the most important portal circulation scavenger in the liver. It is well known that liver injury can induce kupffer cells activation as a major source of inflammatory mediators that can play a role in the pathogenesis of liver diseases (Kolios, Valatas and Kouroumalis, 2006).
http://annalsofrscb.ro 17545 Besides, the evidence of inflammation can be attributed to the fact that OS can augment pro- inflammatory transcriptional regulation by provoking cellular redox signaling pathways, such as NF- 𝜅B, resulting in exaggerated OS through inflammatory cells recruitment and cytokines generation such as TNF-𝛼 and IL-1β, creating another vicious cycle that promotes the pathogenesis of liver disease (Li et al., 2016).
Concerning testicular histopathological results, it has been reported that in testis, 5-HT can induce vasoconstriction which in turn can reduce testicular blood flow that may consequently affect the availability of gonadotropins, oxygen and other nutrients (Ayala et al., 2018), as well as it may explain the demonstrated decline in germ cells count. In addition, it has been established that fluoxetine has a spermicidal action through its binding to sulfhydryl groups in the sperm plasma membrane (Kumar et al., 2006), which can confirm the germ cells degeneration and vacuolations.
While for amitriptyline, a histological study by Tousson et al (2018) (Tousson et al., 2018) revealed that chronic administration of amitriptyline associated with disturbed seminiferous tubules, decrease in the number of spermatogenic cells and moderate vacuolar degenerative changes in the testicular tissues of rats.
Furthermore, the sexual disorders and male infertility induced by amitriptyline have been also proven in the studies of Afify et al (2010) (Afify et al., 2010) and Tousson et al (2018) (Tousson et al., 2018), who both reported that amitriptyline administration caused disturbances of sexual hormones, particularly testosterone, evidenced by impaired spermatogenic cycle at tissue level, which clearly supports the germ cells degeneration and vacuolations that have been shown in the present work.
The present histopathological examination with such interconnected evidences of apoptosis, inflammatory cells infiltration and increased number of kupffer cells provides a pattern for explaining the mechanism of induced hepatic and testicular toxicities that clearly supports the other findings in the context of OS, which are in accordance with the results gathered from the previously mentioned studies.
Conclusion
In the light of the findings presented herein, the present study concludes that the four weeks administration of fluoxetine and amitriptyline can induce oxidative damage in hepatic and testicular tissues of adult male rats, which have been confirmed by histopatholgical examinations.
Therefore, both drugs must be prescribed under careful medical supervision, and a parallel administration of antioxidant agent is recommended.
Further studies should be performed on toxicities of fluoxetine and amitriptyline at different doses with longer treatment periods, to determine their safest doses and durations. More light needed to be shed on the exact molecular mechanisms behind their oxidative stress potentials.
References:
[1] Afify, M. et al. (2010) ‘Differential effects of amitriptyline treatment on testicular and liver functions in adult male rats’, New York Science Journal, 3(3), pp. 10–18.
[2] Agarwal, A. et al. (2014) ‘Effect of oxidative stress on male reproduction’, The world
http://annalsofrscb.ro 17546 journal of men’s health, 32(1), pp. 1–17.
[3] Ali, A. M. and Hendawy, A. O. (2018) ‘So, antidepressant drugs have serious ad‐verse effects, but what are the alternatives’, Nov Appro Drug Des Dev, 4(3), p. 555636.
[4] Ayala, M. E. et al. (2018) ‘Fluoxetine treatment of prepubertal male rats uniformly diminishes sex hormone levels and, in a subpopulation of animals, negatively affects sperm quality’, Reproduction, Fertility and Development. CSIRO, 30(10), pp. 1329–
1341.
[5] Bancroft, J. D. and Gamble, M. (2008) Theory and practice of histological techniques. 6th edn. Livingstone: Churchill Elsevier health sciences.
[6] Bandegi, L. et al. (2018) ‘Effects of antidepressants on parameters, melondiadehyde, and diphenyl-2-picryl-hydrazyl levels in mice spermatozoa’, International Journal of Reproductive BioMedicine. Shahid Sadoughi University of Medical Sciences and Health Services, 16(6), pp. 365–372.
[7] Basol, N. and Erbas, O. (2016) ‘The effects of diltiazem and metoprolol in QTc prolongation due to amitriptyline intoxication’, Human & experimental toxicology.
SAGE Publications Sage UK: London, England, 35(1), pp. 29–34.
[8] Bataineh, H. N. and Daradka, T. (2007) ‘Effects of long-term use of fluoxetine on fertility parameters in adult male rats’, Neuroendocrinology letters. [Weinheim, Ger.] Edition Medizin., 28(3), pp. 321–325.
[9] Bautista-Ferrufino, M. R. et al. (2011) ‘Amitriptyline induces coenzyme Q deficiency and oxidative damage in mouse lung and liver’, Toxicology letters. Elsevier, 204(1), pp. 32–
37.
[10] Beauchamp, C. and Fridovich, I. (1971) ‘Superoxide dismutase: improved assays and an assay applicable to acrylamide gels’, Analytical biochemistry. Elsevier, 44(1), pp. 276–
287.
[11] Bet, P. M. et al. (2013) ‘Side effects of antidepressants during long-term use in a naturalistic setting’, European Neuropsychopharmacology. Elsevier, 23(11), pp. 1443–
1451.
[12] Chee, K. et al. (2015) ‘International study on antidepressant prescription pattern at 40 major psychiatric institutions and hospitals in A sia: A 10‐year comparison study’, Asia‐Pacific Psychiatry. Wiley Online Library, 7(4), pp. 366–374.
[13] Cichoż-Lach, H. and Michalak, A. (2014) ‘Oxidative stress as a crucial factor in liver diseases’, World journal of gastroenterology: WJG. Baishideng Publishing Group Inc, 20(25), pp. 8082–8091.
[14] Deidda, A. et al. (2017) ‘Interstitial lung disease induced by fluoxetine: Systematic review of literature and analysis of Vigiaccess, Eudravigilance and a national pharmacovigilance database’, Pharmacological research. Elsevier, 120, pp. 294–301.
[15] Gupta, S., Nihalani, N. and Masand, P. (2007) ‘Duloxetine: review of its pharmacology, and therapeutic use in depression and other psychiatric disorders’, Annals of Clinical Psychiatry. Taylor & Francis, 19(2), pp. 125–132.
[16] Hines, R. N. et al. (2004) ‘NTP‐CERHR Expert Panel Report on the reproductive and developmental toxicity of fluoxetine’, Birth Defects Research Part B: Developmental and Reproductive Toxicology. Wiley Subscription Services, Inc., A Wiley Company Hoboken, 71(4), pp. 193–280.
[17] Inkielewicz-Stępniak, I. (2011) ‘Impact of fluoxetine on liver damage in rats’, Pharmacological Reports. Springer, 63(2), pp. 441–447.
[18] Jilani, K. et al. (2013) ‘Fluoxetine induced suicidal erythrocyte death’, Toxins.
Multidisciplinary Digital Publishing Institute, 5(7), pp. 1230–1243.
[19] Kampa, J. M. et al. (2020) ‘Mass spectrometry imaging reveals lipid upregulation and bile acid changes indicating amitriptyline induced steatosis in a rat model’, Toxicology
http://annalsofrscb.ro 17547 letters. Elsevier, 325, pp. 43–50.
[20] Kolios, G., Valatas, V. and Kouroumalis, E. (2006) ‘Role of Kupffer cells in the pathogenesis of liver disease’, World journal of gastroenterology: WJG. Baishideng Publishing Group Inc, 12(46), pp. 7413–7420.
[21] Kumar, V. S. et al. (2006) ‘The spermicidal and antitrichomonas activities of SSRI antidepressants.’, Bioorganic & medicinal chemistry letters, 16(9), pp. 2509–2512.
[22] Lee, J., Giordano, S. and Zhang, J. (2012) ‘Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling’, Biochemical Journal. Portland Press Ltd., 441(2), pp. 523–540.
[23] Li, S. et al. (2016) ‘Insights into the role and interdependence of oxidative stress and inflammation in liver diseases’, Oxidative medicine and cellular longevity. Hindawi, 2016.
[24] Moreno-Fernández, A. M. et al. (2008) ‘Cytotoxic effects of amitriptyline in human fibroblasts’, Toxicology. Elsevier, 243(1–2), pp. 51–58.
[25] Ohkawa, H., Ohishi, N. and Yagi, K. (1979) ‘Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction’, Analytical biochemistry. Academic Press, 95(2), pp.
351–358.
[26] Paget, G. E. and Barnes, J. M. (1964) ‘Toxicity tests’, in Laurence DR, Bacharach AL (ed.) Evaluation of drug activities: pharmacometrics. London: Academic Press, pp. 160–
162.
[27] Pilania, M. et al. (2013) ‘Elderly depression in India: An emerging public health challenge’, The Australasian medical journal. Australasian Medical Journal, 6(3), p. 107.
[28] Rajamani, S. et al. (2006) ‘Drug‐induced long QT syndrome: hERG K+ channel block and disruption of protein trafficking by fluoxetine and norfluoxetine’, British journal of pharmacology. Wiley Online Library, 149(5), pp. 481–489.
[29] Rastogi, R. P. and Sinha, R. P. (2009) ‘Apoptosis: molecular mechanisms and pathogenicity’, EXCLI Journal, 8, pp. 155–181.
[30] Safarinejad, M. R. (2008) ‘Sperm DNA damage and semen quality impairment after treatment with selective serotonin reuptake inhibitors detected using semen analysis and sperm chromatin structure assay’, The Journal of urology. Elsevier, 180(5), pp. 2124–
2128.
[31] ‘SAS/STAT Users Guide for Personal Computer. Release 9.13’ (2010) SAS Institute, Inc., Cary, N.C., USA.
[32] Sorodoc, V. et al. (2013) ‘Cardiac troponin T and NT-proBNP as biomarkers of early myocardial damage in amitriptyline-induced cardiovascular toxicity in rats’, International journal of toxicology. SAGE Publications Sage CA: Los Angeles, CA, 32(5), pp. 351–357.
[33] Souza, M. E. J. et al. (1994) ‘Effect of fluoxetine on rat liver mitochondria’, Biochemical pharmacology. Elsevier, 48(3), pp. 535–541.
[34] Tamblyn, R. et al. (2019) ‘Multinational comparison of new antidepressant use in older adults: a cohort study’, BMJ open. British Medical Journal Publishing Group, 9(5), p.
e027663.
[35] Tiller, J. W. G. (2013) ‘Depression and anxiety’, The Medical Journal of Australia, 199(6), pp. S28–S31.
[36] Tousson, E. et al. (2018) ‘Biochemical and immunocytochemical studies of the testicular alteration caused by Amitriptyline in adult male rat’, Journal of Bioscience and Applied Research, 4(4), pp. 418–424.
[37] Wang, Z. et al. (2016) ‘Oxidative stress and liver cancer: etiology and therapeutic targets’, Oxidative medicine and cellular longevity. Hindawi, 2016.
