Research Article

Functional and Histological Signs of Early Detection of Liver and Kidney Stress Under the Influence of Tramadol Oral Administration in Male Rats

Hawraa F.H. Alowaid1*, Afrah Adil Hassan1, Majdy Faisal Majeed2

1College of Dentistry, University of Misan, Iraq; 2Department of Anatomy and Histology, College of Veterinary Medicine, University of Basrah, Iraq.

Received | March 20, 2024; Accepted | April 11, 2024; Published | June 27, 2024

*Correspondence | Hawraa F.H. Alowaid, College of Dentistry, University of Misan, Iraq; Email: [email protected]

Citation | Alowaid HFH, Hassan AA, Majeed MF (2024). Functional and histological signs of early detection of liver and kidney stress under the influence of tramadol oral administration in male rats. Adv. Anim. Vet. Sci., 12(8):1556-1562.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/12.8.1556.1562

ISSN (Online) | 2307-8316

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

All around the world, Addiction to drugs is a social and health problem, one of the most widely abused medicines is analgesics, particularly opioids (Stephen et al., 2011). Tramadol is a synthetic centrally-acting analgesic that has similar effects to codeine but is ten times less expensive than morphine (Marquardt and Albertson, 2005). Tramadol has several applications, however, it is most commonly employed in the treatment of moderate to severe acute or chronic pain (Nossaman et al., 2010). Repeated TR treatment may cause toxic metabolites to build up in the body and reduce TR clearance, raising the risk of TR poisoning (Tjäderborn et al., 2007). Especially in high doses, the harmful consequences of TR are anticipated during long-term therapy (Watson et al., 2004). The oral bioavailability of tramadol is high, ranging from 70 to 80%. After an oral dose, peak blood levels are attained in around 2 hours (Stefan and Armin, 2004). The cytochrome p450 enzyme in the liver breaks it down, and the kidneys are used to eliminate the byproducts. O-desmethyl-tramadol, which is bio-transformed into it in the liver and is 2 to 4 times more effective than tramadol, is an active chemical (Halling and Brosen, 2008). The liver and kidney are in charge of controlling tramadol metabolism and excretion (Al-Mashhadane et al., 2019). As a result, during its metabolism, it may cause hepatotoxicity and nephrotoxicity (Atici et al., 2005). Generally, due to the liver’s crucial involvement in drug metabolism, almost every drug has been linked to hepatotoxicity (Kavita et al., 2018). Drugs’ metabolites, which are excreted by the kidneys, may also induce cellular damage that results in renal failure and may be more active or dangerous than the original drug (Alexander and John 2021). The current study aimed to assess the negative effect of chronic use of tramadol on the liver and kidney, and its relationship with oxidative stress and functional impairment in a rat model. Since tramadol is a commonly used analgesic and can cause addiction when misused, so our information results might be used to create new preventive and therapeutic approaches to combat this problem.

Materials and Methods

Standard solution and single-administration toxicity

Tramadol tablets that each included100 mg of tramadol hydrochloride were provided by Dimidics Company, (Kingdom of Saudi Arabia), A standard solution of tramadol was prepared by dissolving in 100mg/5ml of normal saline to create a solution containing 20 milligrams per 1ml and given group L and H 1.5 and 0.75 ml/kg/day as a single dose by oral gavage, which represents 1/10 and 1/20 of LD50 (Matthiesen et al.,1998).

Experimental design

The study protocols were approved by the Scientific Research Ethics Committee of the Faculty of Veterinary Medicine at Basrah University after the committee took into account the guidelines set forth by the Institutional Animal Care and Use Committees (IACUC) Center for Animal Care. A total of male rats (n= 15) weighing 225±25g, were kept in a controlled environment with a 12-hour cycle of darkness and light at 25±1°C, 48% relative humidity. Rats had unlimited access to tap water and were fed a typical rodent pellet chow diet. Rats were randomly divided according to Essam et al. (2015) into three experimental groups (n= 5): (C) group as control: (2.5ml/kg/day of normal saline), L group (15mg/kg/day of TML), and H group (30mg/kg/day of TML). On day 30, the animals were anesthetized and blood, liver, and kidney samples were collected.

Clinical signs

During the course of the experiment, no deaths were reported using the tramadol medication concentrations employed in this investigation. Comparing drug-treated rats to control rats, the former displayed reduced activity and a decline in feed initiation rates.

Serum enzymes and preparation of tissue homogenate

The biochemical characteristics for biological liver function indicators of male rats serum, Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), and alkaline phosphatase (ALP) enzymes activities were evaluated in all groups by collecting blood into coagulant tubes and centrifuging at 3500 rpm /10 min. The serum was isolated (stored at -20 degrees Celsius for analysis). The enzyme activity of (ALT) and (AST) was determined in IU/l using the IFCC method (Vassault, 1986). While, (ALP) enzyme is evaluated in mg/100 ml using the pNPP Kinetic technique (Bowers and McCommb, 1972). While, the histochemical measurements in the liver and kidney tissues, it was treated as follows: To create 10% homogenates, liver and kidney tissues were cut, weighed, and minced into minute pieces before being homogenized in 9 ml of ice-cold 0.05 mM potassium phosphate buffer (pH 7.4) with a glass homogenizer. The homogenates were centrifuged for 15 minutes at 4°C at 6000 rpm, and the supernatant was used to test malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), and catalase (CAT). Furthermore, the levels of MDA, NO, CAT, and SOD in liver and kidney tissues were determined using the procedures described by (Mesbah et al., 2004; Vodovotz, 1996; Luck, 1974; Kakkar et al. 1984), respectively.

Histological examination

The liver and kidneys organs of all animals euthanized at the end of the experiment were removed and preserved in a 10% neutral buffered formalin solution. After 72 hours of fixation, the samples were washed under running water for two hours. They were then dehydrated by gradually increasing the alcohol concentration from 70% to 100% over a two-hour period for each step. Xylene was used to clean the samples, and then paraffin wax was applied. Finally, the samples were sliced with a microtome to a thickness of 5 um for each tissue. The stains utilized were hematoxylin and eosin (H and E) (Daniel, 2022). Histological alterations were evaluated using an unpretentious scoring system and classified as (-) none, (+) low modification, (++) moderate modification, and (+++) important modification (Suvarna et al., 2012).

Histopathological evaluation of lesion scores

Histological examinations were used to evaluate the degree of the alterations and the distribution of lesions inside the studied organ, depending on the analysis method (Bernet et al., 1999). the distribution of the lesions was determined by scores (Sc) and an important factor (Fi). Sc was represented distributed the total tissue under analysis as (0= absence - 10% of lesions) of, 1= (11-25% of lesions), 2= (26-50% of lesions) 3 = (51-75% of lesions), and 4= (76-100% of lesions). While Fi specifies how such a change will effect organ function, the following values are assigned to it: 1: denotes a lesion that is quickly reversible and has little histological significance. 2: reversible lesions of moderate to neutral histopathological significance; and 3: lesions with high histolopathological significance. The lesion index was determined as follows for each lesion (I Lesion) as the following: I Lesion= (Sc x Fi) and the lesion organ index (I LOrg) is denoted as I LOrgan equals Σ I Lesion.

Data analysis

presented as mean SE, and statistical significance was determined using one-way ANOVA in the (SPSS), version 18. the program was followed by the least significant difference (LSD). When P0.05, values were considered statistically significant.

Results and Discussion

Administration of the tramadol 15 and 30mg/kg/day (L and H groups) at 30 days led to a significant elevation (P≤ 0.05) of AST, ALT, and ALP serum enzymes concentration compare to the control group (Figure 1A). While we detected a significant increase (P≤ 0.05) in MDA from sampled liver and kidney tissues in L and H groups compare to the C group, the higher values were 10.77 and 7.19mmol/g tissue respectively detected in animals from the H group (Figure 1B). NO concentration showed a Significance difference (P≤ 0.05) among all groups, the higher NO values 112.98 and 94.87mmol/g tissue respectively were detected in the H group (Figure 1C). All tramadol groups showed a lower mean of SOD activity (p≤0.05) in liver and kidney tissue compare to the control group, Absolutely the lowest value in the H group was 37.33 and 30.71u/g at liver and kidney tissue respectively (Figure 1D). Administration of the tramadol to the treatment animals group led to inhibition in CAT activity (p≤0.05) in liver and kidney tissue, and the lower values 0.97 and 0.71mmol/g tissue were shown in the H group respectively (Figure 1E).

Histological data

The light microscopic examination of the hematoxylin and eosin-stained paraffin 5 um sections revealed different types of histological lesions of the liver and kidney, which are perfectly in relation with our proven results about the irregular levels of enzymatic and metabolite biomarkers in blood and tissue.

 

Liver

Liver sections revealed in L and H groups, loss of cord-like parenchymal architecture, acute Inflammation infiltration, necrosis and apoptosis state, also showed a hemorrhage, vein congestion, as well as degeneration and vaculation cytoplasmic, excessive sinusoidal dilatation. In general, these histopathological signs were more severe, and clear in the H group of experimental animals (Figure 2).

Kidney

histopathological sections of L and H groups kidneys were a sensitive tool to detect the toxic effect of an overdose of tramadol (15 and 30 mg/kg/day), respectively. The histological alterations revealed slight to severe in each of the following, degeneration of cytoplasm, necrosis of renal tubular cells, necrosis of renal glomeruli, wider urinary spaces, edema of renal tubular cells, hemorrhage, and vessel congestion, generally the H group reversed severe histological alterations.

Histological changes feature of the liver

The values of total histological alteration scores in the control, L, and H groups are shown in Tables 1 and 2. Significant changes in (I Lesion) occurred in rat liver in conjunction with tramadol overdose. L and H groups revealed Subcellular disarrangements included loss of parenchymal cord-like architecture, degeneration and necrosis of hepatocytes, as well as hemorrhage, Vein congestion, Excessive sinusoidal dilatation, and Infiltration of inflammatory cells, in general, these histopathological signs were more severe and clear in the H group of experimental animals, while represented control group, typical appearance of the hepatocytes and parenchymal architecture.

 

Histological changes feature of the kidney

The values of total histological alteration scores in the control, L, and H groups are shown in Tables 3 and 4. Significant changes in (I Lesion) occurred in rat kidney in conjunction with tramadol overdose. L and H groups revealed the histological alterations, discrepancies between slight to severe histopathological alteration, and as blogged below, degeneration of cytoplasm, necrosis of renal tubular cells, atrophied glomeruli, wider Bowman’s spaces, edema, hemorrhage, and vessel congestion, while the control group’s kidney histopathology examination showed the typical appearance and normal of the glomerulus and renal tubular epithelium in all rats (Figure 3).

 

Table 1: Distribution of the histological damage features in the liver of the experimental groups.

Histological signs	Experimental group
	C	L	H
loss of parenchymal cord-like architecture	-	+	++
Degeneration of hepatocytes	-	+	++
necrosis of hepatocytes	-	+	++
hemorrhage	-	+	+++
Vein congestion	+	+++	+++
inflammatory cells	-	+	++
sinusoidal dilatation	+	++	+++

 

(-) nil or mild histological changes; (+) moderate histological changes; (++) severe histological changes; and (+++) very severe histological changes in the liver architecture.

 

Table 2: Liver alteration index (I Lesion%) of rats groups, scores (Sc) and an important factor (Fi) in the study groups.

Histological signs	Liver lesion index (I Lesion%)
	C		L		H
	Fi	Sc	Fi	Sc	Fi	Sc
loss of parenchymal cord-like architecture	0	0	2	55.5	3	73.6
Degeneration of hepatocytes	1	5	3	60.5	3	80.9
necrosis of hepatocytes	1	5	2	50.8	3	78.9
hemorrhage	0	0	2	33.7	3	63.8
Vein congestion	1	5	3	60.6	3	90.5
inflammatory cells	0	0	3	20.8	3	52.9
I LOrgan= Σ I Lesion	1	15%	3	281.9%	3	440.6%

 

I Lesion= (Sc x Fi) and the lesion organ index (I LOrg is denoted by I LOrgan = Σ I Lesion. (Sc): Distributed and Frequently of lesions. (Fi): reversible lesions, reversible lesions, and irreversible.

 

Table 3: Distribution of the histological damage features in the kidney of the experimental groups.

Histological signs	Experimental group
	C	L	H
degeneration of cytoplasm	+	++	+++
necrosis of renal tubular cells	-	++	+++
atrophied glomeruli	-	+	++
edema	-	+	++
hemorrhage	-	++	++
vessel congestion	+	++	+++
inflammatory cells	-	+	++

 

(-) nil or mild histological changes; (+) moderate histological changes; (++) severe histological changes; and (+++) very severe histological changes in the kidney architecture.

 

Table 4: Kidney alteration index (I Lesion %) of rats groups, scores (Sc) and an important factor (Fi).

Histological signs	Kidney lesion index (I Lesion%)
	C		L		H
	Fi	Sc	Fi	Sc	Fi	Sc
Degeneration of cytoplasm	0	0	2	55.5	3	73.6
Necrosis of renal tubular cells
Atrophied glomeruli	1	5	3	60.5	3	80.9
Edema	1	5	2	50.8	3	78.9
Hemorrhage	0	0	2	33.7	3	63.8
Vessel congestion	1	5	3	60.6	3	90.5
Inflammatory cells	0	0	3	20.8	3	52.9
I LOrgan= Σ I Lesion	1	15%	3	281.9%	3	440.6%

 

I Lesion= (Sc x Fi) and the lesion organ index (I LOrg is denoted by I LOrgan= ΣI Lesion. (Sc): Distributed and Frequently of lesions. (Fi): reversible lesions, reversible lesions, and irreversible lesions.

 

 

To better comprehend the intricate mechanics of histology and its potentially harmful biochemical effects, experimental models of opioid addiction in laboratory rats that simulate a human context are a crucial tool, as well as for developing therapeutic strategies for addictive diseases. Over the last ten years, there has been a considerable increase in the abuse of opioid analgesics and anxiolytics around the world, according to the European Medicines Agency’s Committee for Medicinal Products in 2009 stated that there was a lack of information regarding long-term overdose side effects (Jasim et al., 2023). All antipsychotic and morphine drugs derivatives and in acute doses with prolonged periods of abuse for addicted persons cause hepatotoxicity and nephrotoxicity during its metabolism (Mustafa et al., 2021). The marker of oxidative stress (MDA and NO) significantly increased in the current investigation, dysfunction markers in the liver and kidney (ALT, AST, ALP enzymes), and (SOD and CAT enzymes), and histopathological markers, were evident in the overdose oral administration of TML-treated, on liver and kidneys. These data indicate that oxidative stress and dysfunction, play a crucial role in histopathogenesis. tramadol is classified as a drug category C by the American Food and Drug Administration (AFDA), and it is not recommended because of its effects on kidney, liver, or stomach disorders, mental illness, or depression (Kallen, 2015). Our findings suggest that tramadol causes oxidative stress in the tissues of the liver and the kidney, it is consistent with (Costa et al., 2013) and (Abdelraouf et al. 2015). When the liver breaks down different substances, it is known to produce certain metabolites; the toxic metabolites are subsequently packaged for elimination. our results were outcomes resemble those reported by (Nna et al., 2015). Who discovered that the rats in the treatment group had significantly greater blood AST and ALT activity after receiving oral tramadol for 8 weeks. Our results are also came in accordance with the recorded data (Samy et al., 2017). Who observed that using tramadol for 63 days caused a noticeable rise in MAD and NO levels in brain tissue, (Elwy and Tabl, 2014). Which demonstrated a significant reduction in the SOD, CAT, and GSH activities in the liver tissue as compared to control rats. Additionally, (Nafea et al., 2016). demonstrated that abuse of tramadol for a month caused a considerable rise in MDA and a fall in antioxidant (CAT) activity. Ahmed and Kurkar (2014) also had similar findings in testicular tissue, reporting that tramadol enhanced testicular levels of No while dramatically decreasing enzymatic antioxidant activity. ROS renders all antioxidant enzymes inactive, generally, Nitric oxide (NO) and malondialdehyde (MDA) levels are frequently utilized as a biomarker of free radical-mediated lipid peroxidation because they show how much cellular harm that free radicals have indirectly caused (Nematollah et al. 2017). Our histopathological results of overdose tramadol toxicity was confirmed in liver and kidney tissue, This may be explained by the fact that the liver is in charge of tramadol’s metabolism and excretion (Shah et al., 2013). While the kidneys play a role in the secretion of numerous toxins, they are also prone to release large amounts of free radicals, which in turn contribute to high levels of oxidative stress and kidney damage (Ghosh et al., 2010). Our findings supported the findings of (Al-Mashhadane et al. 2019), which claimed that tramadol usage over an extended period of time causes localized vessel congestion, necrosis hepatocyte, and cytoplasmic degeneration in rats. Because the kidneys are involved in the release of numerous toxins, they are prone to producing significant numbers of free radicals, which enhance oxidative stress and cause kidney disease (Ghosh et al., 2010). Most of the histopathological findings obtained in our study were similar to what was reported (İsa et al., 2019). Which confirmed that given that tramadol and its metabolites are eliminated through the kidneys, there is a chance that they could cause nephrotoxicity and histological changes. Overall, the current study’s findings point to oxidative stress and free radicals as processes implicated in the histopathological of TML-induced hepatocytes and renal cell impairment, so more research is needed to create preventive/ therapeutic measures based on these findings.

Acknowledgement

Data gathering, experimental setup, and data visualization were carried out by Majdy Faisal Majeed and Alaa Hussein Sadoon. Dr. Majdy Faisal Majeed worked on data visualization, formal analysis, and research topics. The creation and modification of the paper draft involved both writers. The final version of the manuscript, as well as the draft(s), were all read and approved by all authors.

Author’s Contribution

HFHA: Writing and analyzing the physiological results of the research, AAH analyzing and interpreting biochemical results. MFM design experiments and analysis of data statistically and interpret histopathological results.

Ethical clearance

This experiment followed the animal ethics committee’s set rules, which were based on internationally accepted principles for laboratory animal use and care.

Additional information

There is no extra information for this paper.

Abbreviations

Tramadol (TML), High dose (H), Low dose (L), superoxide dismutase (SOD), catalase (CAT), Malondialdehyde (MDA), Nitric oxide (NO), Scores (Sc), important factor (Fi), Alanine aminotransferase enzymes (ALT), Aspartate aminotransferase enzymes (AST), Alkaline phosphatase enzymes (ALP), Institutional Animal Care and Use Committees (IACUC).

Conflict of interest

The authors state that they have no known competing financial or personal interests that could appear to have impacted the work presented in this paper.

References

Abdelraouf A, Elmanama AA, Abu Tayyem NE, Essawaf HN, Hmaid IM (2015). Tramadol-induced liver and kidney toxicity among abusers in Gaza Strip, Palestine. Jordan J. Biol. Sci., 8(2): 133–137. https://doi.org/10.12816/0027559

Ahmed MA, Kurkar A (2014). Effects of opioid (tramadol) treatment on testicular functions in adult male rats: The role of nitric oxide and oxidative stress. Clin. Exp. Pharmacol. Physiol., 41: 317-323. https://doi.org/10.1111/1440-1681.12213

Alexander M, John RA (2021). Drug therapies affecting renal function: An overview. Cureus, 13(11): 2-11.

Al-Mashhadane FA, Ismail HK, Al-Saidya AM (2019). Histopathological effects of chronic use of tramadol on liver and kidney in sheep. Model J. Pharm. Sci. Res., 11(6): 2208-2212.

Atici, S., Cinel, I., Cinel, L., Doruk, N., Eskandari, G., & Oral, U. (2005). Liver and kidney toxicity in chronic use of opioids: an experimental long term treatment model. J. Biosci., 30:245-252.

Bernet D, Schmidt H, Meier W, Burkhardt-Holm P, Wahli T (1999). Histopathology in fish: Proposal for a protocol to assess aquatic pollution. J. Fish Dis., 22: 25-34. https://doi.org/10.1046/j.1365-2761.1999.00134.x

Bowers GN, McCommb RB (1972). Recommendation of the German society for clinical chemistry. Z. Clin. Chem. Biol., 10: 182.

Costa PF, Nunes N, Belmonte EA, Moro JV, Lopes PC (2013). Hematologic changes in propofol-anesthetized dogs with or without tramadol administration. Arq. Bras. Med. Vet. Zootec., 65: 1306-1312. https://doi.org/10.1590/S0102-09352013000500007

Daniel C (2022). Histological staining and its method. J. Interdiscip. Histopath., 10(9): 1-2.

Elwy AM, Tabl G (2014). Effects of chronic usage of tramadol, acetaminophen and tramacet on some biochemical and immunological changes in male rats. J. Drug Res. Energy, 35(1): 63-71.

Essam HM, Sahar IY, Safaa ARM (2015). Parenchymatous toxicity of tramadol: Histopathological and biochemical study. J. Alcohol Drug Depend., 3(5): 2-6.

Ghosh JJ, Das P, Manna, Sil PC (2010). Acetaminophen induced renal injury via oxidative stress and TNF-alpha production: Therapeutic potential of arjunolic acid. Toxicology, 268: 8-18. https://doi.org/10.1016/j.tox.2009.11.011

Halling J, Weihe P, Brosen K (2008). CYP2D6 polymorphism in relation to tramadol metabolism: A study of Faroese patients. Therap. Drug. Monitor., 30: 271-275. https://doi.org/10.1097/FTD.0b013e3181666b2f

İsa A, Muhammet EG, Mustafa A, Yavuz Y (2019). Murat ozturk4 histopathological findings in patients with lipoid proteinosis. Turk. J. Dermatol., 13(3): 99-102. https://doi.org/10.4103/TJD.TJD_8_19

Jasim NH, Mohammed YJ, Alahmed JAS, Majeed MF (2023). Methylphenidate hydrochloride overdose induces liver damage in rats: The evaluation of histopathology and some antioxidant enzymes biomarkers. Adv. Anim. Vet. Sci., 11(6): 933-938. https://doi.org/10.17582/journal.aavs/2023/11.6.933.938

Kakkar P, Das B, Viswanathan PN (1984). A modified spectrophotometric assay of superoxide dismutase. Indian J. Biochem. Biophys., 21: 130-132.

Kallen MR (2015). Use of tramadol in early pregnancy and congenital malformation. Risk Reprod. Toxicol., 58: 246–251. https://doi.org/10.1016/j.reprotox.2015.10.007

Kavita G, Mohd RR, Nishant R, Arunabha R (2018). Hepatotoxicity: Its mechanisms, experimental evaluation and protective strategies. Am. J. Pharmacol., 1(1): 2-9.

Luck H (1974). Estimation of catalase. In: Methods in enzymatic analysis. 2nd ed, New York. Bergmeyer. Academic Press, pp. 885-890.

Marquardt KA, Alsop JA, Albertson TE (2005). Tramadol exposures reported to statewide poison control system. Ann. Pharmacother., 39(6): 1039–1044. https://doi.org/10.1345/aph.1E577

Matthiesen T, Wöhrmann T, Coogan T, Uragg PH (1998). The experimental toxicology of tramadol: An overview. Toxicol. Lett., 95(1): 63-71. https://doi.org/10.1016/S0378-4274(98)00023-X

Mesbah L, Soraya B, Narimane S, Jean PF (2004). Protective effect of flavonides against the toxicity of vinblastine cyclophosphamide and paracetamol by inhibition of lipid peroxydation and increase of liver glutathione. Haematology, 7(1): 59-67.

Mustafa SG, Dhuha AK, Majdy FM (2021). Histopathological changes in the liver and kidney in rabbits exposed to overdose of Olanzapine drug. Biochem. Cell. Arch., 21: 1853-1857.

Nafea OE, El-Khishin IA, Awad OA, Mohamed DA (2016). A study of the neurotoxic effects of tramadol and cannabis in adolescent male albino rats. Int. J. Sci. Rep., 2(7): 143-154. https://doi.org/10.18203/issn.2454-2156.IntJSciRep20162164

Nematollah A, Mahmoud B, Arash K, Mahmoud R (2017). The impact of oxidative stress on testicular function and the role of antioxidants in improving it: A review. J. Clin. Diagn. Res., 11(5): 1-5.

Nna VU, Akpan UP, Okon VE, Atangwho IJ (2015). Hepatotoxicity following separate administration of two phosphodiesterase-5 inhibitors (sildenafil and tadalafil) and opioid (tramadol); evaluation of possible reversal following their withdrawal. J. Appl. Pharm. Sci., 5(8): 105-113. https://doi.org/10.7324/JAPS.2015.50817

Nossaman VE, Ramadhyani U, Kadowitz PJ, Nossaman BD (2010). Advances in perioperative pain management: Use of medications with dual analgesic mechanisms, tramadol and tapentadol. Anesthesiol. Clin., 28: 647–666. https://doi.org/10.1016/j.anclin.2010.08.009

Samy AH, Samir ALA, Hany KI (2017). Neurodegeneration and oxidative stress induced by tramadol administration in male rats: The effect of its withdrawal. Benha Vet. Med. J., 33(2): 149-159. https://doi.org/10.21608/bvmj.2017.30017

Shah NH, Thomas E, Jose R, Peedicayil J (2013). Tramadol inhibits the contractility of isolated human myometrium. Auton. Autacoid. Pharmacol., 33: 1-5. https://doi.org/10.1111/aap.12003

Stefan G, Armin S (2004). Clinical pharmacology of tramadol. Clin. Pharmacol. Kinet., 43(13): 879-923. https://doi.org/10.2165/00003088-200443130-00004

Stephen FB, Ryan AB, Theresa AC, Taryn MD, Simon HB (2011). Abuse risks and routes of administration of different prescription opioid compounds and formulations. Harm. Redu. J., 8 (29): 2-17. https://doi.org/10.1186/1477-7517-8-29

Suvarna M, Anuradha C, Kumar KK, Sekhar PC, Chandra KLP, and Reddy BR (2012). Cytomorphometric analysis of exfoliative buccal cells in type II diabetic patients. J. Dr. NTR Univ. Health Sci.1(1): 33-37.

Tjäderborn M, Jönsson AK, Hägg S, Ahlner J (2007). Fatal unintentional intoxication with tramadol during 1995-2005. Forensic Sci. Int., 173: 107-111. https://doi.org/10.1016/j.forsciint.2007.02.007

Vassault A (1986). Protocol de validation de techniques. Ann. Biol. Clin., 44: 686.

Vodovotz Y (1996). Modified microassay for serum nitrite and nitrate. Biol. Tech., 20: 390-394. https://doi.org/10.2144/19962003390

Watson W, Litovitz TL, Klein-Schwartz W, Rodgers GC, Youniss J, Reid N, Borys D 2004. Toxic exposure surveillance system. Annual report of the American Association of Poison Control Centers (Ultram). Am. J. Emerg. Med., 22: 335–404. https://doi.org/10.1016/j.ajem.2004.06.001