|Year : 2016 | Volume
| Issue : 4 | Page : 1122-1129
Biochemical, histopathological, and immunohistochemical changes on the liver of adult albino rats due to dependence on tramadol, diazepam, and their combination
Samy M Badawi1, Samy A Hammad1, Safaa A Amin1, Azza W Zanaty1, Hayam A Aiad2, Reham H Mohamed MSc 1
1 Department of Forensic Medicine and Clinical Toxicology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||23-Nov-2014|
|Date of Acceptance||06-Jan-2015|
|Date of Web Publication||21-Mar-2017|
Reham H Mohamed
Department of Forensic Medicine and Clinical Toxicology, Faculty of Medicine, Menoufia University, Menoufia Governorate, 32511
Source of Support: None, Conflict of Interest: None
This study aimed to evaluate the toxic effects of the dependence of tramadol, diazepam, and their combination on the liver of adult albino rats.
Nowadays, tramadol is the most common drug of abuse; it has been associated with a wide range of drug abuse such as benzodiazepines. The combination of tramadol and benzodiazepines has serious detrimental effects on the liver.
Materials and methods
In this study, 40 adult albino male rats weighing 180–200 g were obtained from the breeding animal house in Menoufia University; they were divided into four groups: group 1 (control) received 1 ml/day normal saline (0.9%) by oral tube, group 2 (tramadol dependent) received increasing doses of tramadol by oral tube for 1 month, group 3 (diazepam dependent) received increasing doses of diazepam by oral tube for 1 month, and group 4 (combination of both drugs) received increasing doses of tramadol and diazepam by oral tube for 1 month. At the end of the experimental period blood samples were taken from all groups for evaluation of the liver function, after which the rats were killed. In addition, histopathological and immunohistochemical examinations of the liver were carried out.
Tramadol and diazepam dependence affect the liver function parameters, as alanine transaminase and aspartate transaminase were significantly elevated. Liver enzymes were much affected by their combination; these results were proved by the histopathological and immunohistochemical examination of the liver tissue.
Tramadol or diazepam dependence for a long time might affect the hepatic cells and the combination of both of them leads to more hepatotoxic effect. Therefore, it is recommended that tramadol or diazepam should be taken only with the prescription of doctor and that self-medication of these drugs may be hazardous.
Keywords: dependence, diazepam, liver, tramadol
|How to cite this article:|
Badawi SM, Hammad SA, Amin SA, Zanaty AW, Aiad HA, Mohamed RH. Biochemical, histopathological, and immunohistochemical changes on the liver of adult albino rats due to dependence on tramadol, diazepam, and their combination. Menoufia Med J 2016;29:1122-9
|How to cite this URL:|
Badawi SM, Hammad SA, Amin SA, Zanaty AW, Aiad HA, Mohamed RH. Biochemical, histopathological, and immunohistochemical changes on the liver of adult albino rats due to dependence on tramadol, diazepam, and their combination. Menoufia Med J [serial online] 2016 [cited 2020 May 26];29:1122-9. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/1122/202526
| Introduction|| |
Drug abuse is a problem that has been increasing immensely among our society today. People sometimes feel that they are too bright, too powerful, too much in control to become addictive, but addiction can trap anyone .
Tramadol is used as a centrally acting analgesic and was considered a safe drug devoid of many serious adverse effects of traditional opioids. However, toxicity and abuse potentiality of tramadol have been reported. Tramadol produces analgesia by the synergistic action of the parent drug and its O-desmethylated metabolites . It acts as a weak opioid agonist and as an inhibitor of the neuronal reuptake of the neurotransmitters norepinephrine and serotonin (5-HT) – both neurotransmitters are involved in the mechanism of drug addiction .
Tramadol is rapidly absorbed orally; a peak concentration is detected 2–3 h after an oral dose. It has extensive tissue distribution. Thirty percent of the drug is excreted through the kidneys in an unchanged manner. Elimination half-life is 5–6 h, whereas the remaining is metabolized in the liver by N-demethylation and O-demethylation, followed by conjugation with glucuronic acid and sulfate. The active metabolite, O-desmethyl tramadol shows higher affinity for the µ-opioid receptors and has twice the analgesic potency of the parent drug .
Benzodiazepines (BZDs) are sedative-hypnotic agents that are the most frequently prescribed class of psychotropic drugs. BZDs, such as diazepam, are commonly used for their anxiolytic and sedative effects by their action on the γ-aminobutyric acid A receptor complex, present in the central nervous system . In addition to the central receptors described for BZD, peripheral-type binding sites had been identified in liver cells, endocrine steroidogenic tissues, and immune cells .
Every drug has been associated with hepatotoxicity almost certainly due to the pivotal role of the liver in drug metabolism. Hepatic metabolism is a mechanism that converts drugs and other compounds into products that are more easily excreted. A metabolite may have higher activity and/or greater toxicity than the original drug .
There are many reports regarding the induction of apoptosis by major components of morphine, codeine, noscapine, and papaverine  that indicate that opiates probably play an important role in brain and liver cells apoptosis, therefore leading to neurotoxicity and hepatotoxicity .
The molecular mechanisms of opioid-induced apoptosis have not been established yet. Various key proteins are involved in the regulation of programmed cell death . The p53 tumor suppressor protein also plays a central role in cell cycle arrest and apoptosis . P53 is a proapoptotic short-lived and constitutively expressed at low levels in most cell types .
| Materials and Methods|| |
- Tramadol hydrochloride: pure powder, obtained from Sigma Co. for Pharmaceutical and Chemicals (Quesna, Egypt)
- Diazepam: pure powder, obtained from Nile Co. for Pharmaceutical and Chemicals (Cairo, Egypt).
Animals and experimental design
The study was conducted on 40 adult albino male rats weighing 180–200 g that were obtained from the breeding animal house in Menoufia University.
They were left to acclimatize for 1 week. They were housed at room temperature in metallic cages and were kept under constant healthy environmental and nutritional conditions. Animals were kept under a schedule of diurnal lighting conditions (12 h of darkness and 12 h of light); they were fed on ordinary food and housed under standard laboratory conditions.
The maintenance of the animals and the experimental procedures were in accordance with the guiding principles of Ethical Committee of Menoufia University.
Group 1 (the control group)
This group consisted of 10 rats that were kept in the cages without handling to show the normal values of the tested parameters. Each animal received 1 ml/day normal saline (0.9%) by oral tube (a process called gavage) ; throughout the experiment they were kept under the same conditions for 1 month as the rats of groups 2, 3, and 4.
Group 2 (tramadol dependent)
This group consisted of 10 rats. Each animal received tramadol in gradually increasing doses until it reached the dependent dose for 1 month. Dependence was induced by giving the maximal therapeutic dose of tramadol to start with, which was calculated according to Paget's equation . The therapeutic dose for rat weighing 200 g = 18/1000 × adult human therapeutic daily dose (400 mg)  = 7.2 mg. Then the dose was gradually increased by adding the initial calculated therapeutic dose every 3 days till the end of the month. The calculated tramadol doses were delivered orally in normal saline by a curved needle like oral tube that was introduced directly into the stomach (a gavage process) .
Group 3 (diazepam dependent)
This group consisted of 10 rats that received diazepam in gradually increasing doses until it reached the dependent dose in 1 month. Dependence was induced by giving the maximal therapeutic dose of diazepam to start with it, which was calculated according to Paget's equation . The therapeutic dose for rat weighing 200 g = 18/1000 × adult human therapeutic daily dose (40 mg)  = 0.72 mg. Then the dose was gradually increased by adding the initial calculated therapeutic dose every 3 days till the end of the month. The calculated diazepam doses were delivered as suspension in normal saline and given orally to each animal by a curved needle-like oral tube that was introduced directly into the stomach (a gavage process) .
Group 4 (tramadol and diazepam dependent)
This group consisted of 10 rats that received combined therapeutic oral dose of tramadol = 7.2 mg and diazepam = 0.72 mg in the start. Then the dose was gradually increased by adding the initial calculated therapeutic dose every 3 days till the end of 1 month.
At the end of the experiment, blood samples were collected from rats of all groups from the retro-orbital venous plexus for biochemical serum analysis. After that rats were killed by cervical dislocation and autopsy was carried out where the liver samples were collected.
Biochemical serum analysis
Alanine transaminase (ALT) and aspartate transaminase (AST) were assayed according to Keiding et al. and serum albumin was done by using the quantitative modified bromocresol green colorimetric method of Doumas et al. .
Histopathological and immunohistochemical studies
The liver tissues were taken from the eviscerated rats and fixed in 10% formalin for 24 h, and then processed to obtain paraffin blocks. Sections of 4–6 µm thickness were cut using a microtome and stained with hematoxylin and eosin (H and E) stain by using the method of Stevens and Wilson  and by immunohistochemical staining using anti-p53 stain according to the method of Joyner and Wall .
Data were organized, tabulated, and statistically analyzed using the SPSS Inc. Released 2007. SPSS for Windows, Version 16.0. (SPSS Inc., Chicago, USA). For quantitative data, the mean and SD were calculated. The difference between two means was statistically analyzed using Student's t-test. For comparison of means of more than two groups, the F-test was used. Statistical significance was taken at a P value of less than 0.05.
| Results|| |
Concerning the liver biochemical parameters in [Table 1], AST and ALT were highly significantly increased and the serum albumin was significantly decreased in group 2 compared with group 1. AST and ALT in group 3 were significantly increased compared with group 1, whereas serum albumin was not significantly changed compared with group 1.
|Table 1 Comparison of liver function test between group 1, group 2, and group 3 in experimental albino rats|
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[Table 2] reveals that AST and ALT were highly significantly increased in group 4 as compared with group 2 or group 3, whereas serum albumin was not significantly changed.
|Table 2 Comparison of liver function tests between group 4, group 2, and group 3 in experimental albino rats|
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As per microscopic examination, the liver specimens of rats stained by H and E in group 1 were free of pathological changes [Figure 1]. However, an increase in the prevascular cellular infiltration, marked congestion, and Kupffer cell hyperplasia were observed in almost all rats. Steatosis and marked degeneration of hepatocytes and some apoptotic cells were observed in group 2 [Figure 2]. In group 3 there was congestion of central vein and ballooning degeneration of hepatocytes in zone 1 [Figure 3]. Whereas in group 4, the previously mentioned changes were markedly intensified with severe hemorrhage and marked sinusoidal dilatation and the liver cells were highly degenerated with an area of necroinflammation and Kupffer hyperplasia [Figure 4].
|Figure 1: Photomicrograph of a section of the liver in group 1 (control) showing normal hepatocytes cells between the portal tract and the central vein (H and E, ×200).|
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|Figure 2: Photomicrograph of the liver sections in group 2 (tramadol dependent) (a) showing inflammatory cellular infiltration (i), congested blood sinusoids (c), steatosis (s), and degenerated cells (d) (H and E, ×200); (b) showing Kupffer cell hyperplasia (red arrow), degenerated hepatocytes, and apoptotic cells (black arrow) with area of necrosis (green arrow) (H and E, ×400).|
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|Figure 3: Photomicrograph of the liver sections in group 3 (diazepam dependent) (a) showing congestion of central vein and ballooning degeneration of some of hepatocytes cells (H and E, ×200); (b) showing marked steatosis and congestion with some apoptotic cells (H and E, ×400).|
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|Figure 4: Photomicrograph of the liver sections in group 4 (tramadol and diazepam dependent) (a) showing hemorrhage (h) and sinusoidal dilatation (s), vasculation (v) and the cells are highly degenerated with pyknotic nuclei and cell ghost (g) (H and E, ×200); (b) showing confluent necroinflammation (NI), degenerated cells with pyknotic nuclei (green arrow), and congestion (red arrow) in sinusoids (H and E, ×400).|
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Microscopic examination of the liver specimens of the rats stained by p53 in group 1 showed negative immune reaction for p53 staining. In group 2 and group 3, there were positive expression of p53 reaction, which was more described in group 2 than in group 3. Whereas in group 4, there was a marked increase in the expression of p53 reaction [Figure 5].
|Figure 5: Photomicrograph of the liver sections stained with p53 immunohistochemical (a) showing negative expression of p53 in group 1 (control); (b and c) showing positive expression of p53 in group 2 and group 3, respectively. (d) Marked increase in the expression of p53-positive apoptotic cells in group 4 compared with groups 1, 2, or 3 (p53 × 200).|
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| Discussion|| |
Nowadays tramadol has become one of the most commonly abused drugs, as an alternative to narcotics due to difficulty of getting the later. Other drugs have been found to be used in combination with tramadol, among which BZDs were the most commonly used .
BZDs are commonly used by opioid abusers to reduce anxiety, reinforce opioid effects, and treat craving and withdrawal symptoms . Moreover, the expert dependent patient learned to prevent the seizures that develop with high doses of tramadol by self-medicating with BZDs . Among BZDs, diazepam is one of the most preferred, prescribed, and thus abused molecules .
The present study was conducted to evaluate the effects of the dependence of tramadol, diazepam, and their combination on biochemical, histopathological, and immunohistochemical changes in the liver of albino rats.
In group 2, AST and ALT were highly significantly increased compared with group 1; this was in agreement with Atici et al. . Increase in the AST level can occur in connection with damages of the heart or skeletal muscle as well as of liver parenchyma; however, liver-specific enzyme ALT has been shown to be only significantly elevated in hepatobiliary disease .
Hepatic metabolism is, first and foremost, a mechanism that converts drugs and other compounds into products that are more easily excreted and that usually have a lower pharmacologic activity than the parent compound. A metabolite may have higher activity or greater toxicity than the original drug .
Being an opioid, tramadol carries all possible risks known from other opiates. Experimental studies have also supported the toxic effects of the chronic use of opioids on the liver .
Serum albumin was significantly decreased in group 2 compared with group 1; this can be explained by the fact that the rat's serum albumin depends on an adequate supply of amino acids for its synthesis and thus on the nutritional status , so this was markedly affected in group 2 because of anorexia, and weight loss; thus malnutrition may result in a low rate of albumin production. This was in agreement with Elyazj et al.  who found lower synthesis of albumin and globulin in the liver in response to chronic tramadol intake. Albumin reflects hepatic function; it is one of the standard liver function tests. The two most influential factors regulating hepatic albumin synthesis are nutritional intake, specifically protein consumption, and illness. Reduced protein consumption slows mRNA synthesis of albumin and results in lower serum levels .
In addition, AST and ALT were significantly increased in group 3 compared with group 1; this might be due to the oxidative tissue damage induced in the liver in response to repeated ingestion of diazepam and the adverse effects of its active metabolites such as desmethyldiazepam. The increase in such marker enzyme activities is mainly due to the leakage of these enzymes from liver cytosol into the blood stream as a result of tissue damage .
This result was in agreement with the studies of Jeboori and colleagues , who showed that diazepam caused increase in liver enzymes activity, and also with Abdelmajeed  who stated that long use of diazepam increases the liver enzyme activity (ALT and AST), which might be due to oxidative tissue damage indicated by the marked elevated activity of a xanthine oxide accompanied by the increased nitric oxide levels in the liver. Bahman et al. reported that repeated oral administration of diazepam significantly elevated serum concentrations of ALT, AST, alkaline phosphates, and lactic dehydrogenase in cats.
Serum albumin in group 3 was not significantly changed compared with group 1; this may be attributed to mild affection on the nutritional status in the diazepam-dependent group.
AST and ALT of the liver function were highly significantly increased in group 4 compared with group 2 or group 3, whereas serum albumin was not significantly changed in group 4 compared with group 2 or group 3. This indicates the more toxic effect of the combination of both drugs on the liver cells and enzymes.
Tramadol and diazepam are mainly metabolized in the liver by the cytochrome P450 family of isozymes . Diazepam is metabolized into desmethyldiazepam, which has a half-life of 36–200 h . The elimination half-life of O-desmethyl tramadol (M1), the major active metabolite of tramadol, is slightly longer than that of tramadol itself. These long-acting metabolites are partial agonists and their accumulation may be the cause of affection of the liver enzymes AST and ALT. In addition, possible cytochrome P450-based interaction between tramadol and diazepam may have a role in liver affection .
Many researches have shown that opioids and BZDs exert significant modulatory effects upon one another, where BZDs may alter the pharmacokinetics of opioids ,. Shah et al. reported that when diazepam was administered 1-h before methadone, an increase in methadone concentrations occurred in hepatic and brain tissues along with decreased urinary and hepatic methadone metabolites. The highest values of liver enzymes shown by the combination of diazepam and tramadol dependence in group 4 indicates the more toxic effect on liver cells.
Regarding histopathological examination of the liver tissue in group 2, the increase in the prevascular cellular infiltration, congestion, and Kupffer cell hyperplasia (observed in almost all liver specimens), steatosis, the increase in degenerated hepatocytes, and apoptotic cells might be explained by direct hepatocellular injury during metabolism or by the effect of its metabolites, in addition to the ischemic effect or mitochondrial toxicity after repeated tramadol intake .
Atici et al. reported severe centrolobular congestion and focal necrosis in the rat liver of the chronic tramadol group, which pointed out the risk for increased hepatic damage due to long-term use of tramadol, which was in line with our findings. In addition, our findings were in agreement with that of the study of Samaka et al. where marked inflammatory cellular infiltrate, fibrosis, and bile duct proliferation were noticed in all cases of the tramadol-dependent group.
Microscopic examination of the liver specimens of group 3 rats showed congestion of central vein, marked steatosis, and ballooning degeneration of hepatocytes cells around the portal tract in zone 1.
These pathological lesions in the liver may be due to drug toxicant and adverse effects of its active metabolites and the accumulative ability of diazepam during repeated administration. The present results with involvement of free radical-mediated pro-oxidative processes in the brain and liver following diazepam administration , suggesting that diazepam has an important role in the development of oxidative stress in the brains and livers of diazepam-treated rats. Moreover, our results were coped with those of Abdelmajeed and colleagues ,.
Histopathologic alterations were obviously increased in group 4 (tramadol and diazepam were used in combination at increasing doses) in the form of severe hemorrhage, marked sinusoidal dilatation, Kupffer cells hyperplasia, and apoptosis and the cells were highly degenerated with an area of necroinflammation, which highlighted the fact that coadministration of both drugs enhances their toxic effects; this was in agreement with the biochemical results.
Immunohistochemical results (apoptosis) supported the histopathological and biochemical changes. There was a negative immune reaction in group 1 in the liver, whereas positive expressions of p53 were present in groups 2, 3, and 4, which was proportionated to the degeneration and death of the cells where there was mild expression in group 3, moderate expression in group 2, and marked expression in group 4.
We propose that apoptosis-inducing effect of tramadol in the liver cells may be related to its inhibition of mitochondrial electron transport chain where the role of mitochondria in apoptosis has been well established .
And the marked expression of positive apoptotic cells with p53 stain in group 4 proves the histopathological and biochemical alterations that were previously reported in this study. In a similar study by Mohamed et al. , conducted to study the combined effect of clonazepam and tramadol on mitochondrial chain, clonazepam alone did not show any inhibitory effect at any level; however, its combination with tramadol boosted its toxic effect especially at high doses. It seems like it acts as synergism for tramadol effect.
These results were in agreement with Asiabanha et al. who found that opium compounds play an important role in brain and liver cells apoptosis, therefore leading to neurotoxicity and hepatotoxicity.
Pathogenesis of drug-induced liver injury (DILI) and hepatic apoptosis includes cell stress, mitochondrial impairment, and specific immune reactions. Parent drugs or their reactive metabolites can cause cell stress to produce cytokines, chemokines, reactive oxygen species, and reactive nitrogen species. Current concepts emphasize the central role of mitochondria, which leads to apoptotic and/or necrotic cell death. Hepatocytes, Kupffer cells, and even endothelial cells all participate in DILI. Inflammatory mediators of the innate immune system determine the outcome of DILI .
The molecular mechanisms of opioid-induced apoptosis have not been established yet. Various key proteins are involved in the regulation of programmed cell death .
There are many reports regarding the induction of apoptosis by major components of morphine, codeine, noscapine, and papaverine , which indicate that opiates probably play an important role in brain and liver cells apoptosis, therefore leading to neurotoxicity and hepatotoxicity .
Tramadol and/or its active metabolite may produce excessive release of reactive oxygen species leading to single-strand or double-strand DNA breaks. The accumulation of DNA strand breaks is a well-established stimulus for p53 activation. In addition, oxidative damage of proteins involved in cell cycle regulation or DNA repair may contribute to accumulating DNA damage and finally to activation of p53, which, in turn, mediates either DNA repair or apoptosis .
In agreement with these findings the present results indicate that chronic use of tramadol or diazepam increased hepatic apoptosis, which increased markedly by their combination.
| Conclusion|| |
In agreement with the biochemical findings, which were supported by the histopathological and immunohistochemical changes in the liver of the treated rat groups, our results pointed out the risk for hepatic tissue damage due to dependence on tramadol or diazepam, which increased markedly by their combination. Therefore, it is recommended that these drugs should be taken only with the prescription of doctor and that self-medication or abuse may be hazardous. Moreover, future studies should be carried out to explore the toxic effect of the long-term use of these drugs on other organs such as the brain, heart, and kidney.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Mattoo SK, Nebhinan N, Kumar BN, Basu D, Kulhara P. Family burden with substance dependence: a study from India. Indian J Med Res 2013; 137
Souza MJ, Martin-Jimenez T, Jones MP, Cox SK. Pharmacokinetics of oral tramadol in red-tailed hawks (Buteojamaicensis). J Vet Pharmacol Ther 2011; 34
Vozarova B, Stefan N, Lindsay SR, Saremi A, Pratley ER, Bogardus C, et al.
High alanine aminotransferase associated with decreased hepatic insulin sensitivity and predicts the development of type 2 diabetes. Diabetes 2002; 51
Samaka R, Girgis N, Shams T. Acute toxicity and dependence of tramadol in albino rats: relationship of nestin and notch 1 as stem cell markers. J Am Sci 2012; 8
Abdelmajeed N. Diazepam-induced oxidative stress in rat different organs. Res J Med Med Sci 2009; 4
Bronstein AC, Spyker DA, Cantilena LR, Green JL, Rumack BH, Giffin SL. Annual Report of the American Association of Poison Control Centers' National Poison Data System (NPDS):26th
Annual Report. Clin Toxicol (Phila) 2009; 47
Atici S, Cinel I, Cinel L, Doruk N, Eskandari G, Oral U. Liver and kidney toxicity in chronic use of opioids: an experimental long term treatment model. J Biosci 2005; 30
Shah NS, Patel VO, Donald AG. Effect of diazepam, desmethylimipramine, and SKF 525-A on the disposition of levo-methadone in mice after single or double injection. Drug Metab Dispos 1979; 7
Asiabanha M, Asadikaram G, Rahnema A, Mahmoodi M, Hasanshahi G, Hashemi M. Chronic opium treatment can differentially induce brain and liver cells apoptosis in diabetic and non-diabetic male and female rats. Korean J Physiol Pharmacol 2011; 15
Sastry PS, Rao KS. Apoptosis and the nervous system. J Neurochem 2000; 74
Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B. A model for p53-induced apoptosis. Nature 1997; 389
Wheatley JL. A gavage dosing apparatus with flexible catheter provides a less stressful gavage technique in the rat. Lab Anim (NY) 2002; 31
Paget GE, Barnes JM. Interspecies dosage conversion scheme in evaluation of results and quantitative application in different species. In: Laurence DR, Bacharach AL, (editors). Evaluation of drug activities: pharmacometrics
. London and New York; Academic Press; 1964. 160–162.
Ali AH, Zinad KH. Histopathological changes and immunosuppression induce by diazepam in mice. Al-Qadisiya J Vet Med Sci 2014; 13
Keiding R, Horder M, Gerhardt W. Recommended methods for determination of four enzymes in blood. Scand J Clin Lab Investig 1974; 33
Doumas BT, Watson WA, Biggs HG. Albumin standard and the measurement of serum albumin with bromocresol green. Clin Chem Acta 1971; 31
Stevens A, Wilson IG. The haematoxylin and eosin. In: Bancroft JD, Stevens A, Turner DR, editors. Theory and practice of histological techniques
ed. New York: Churchill Livingstone; 1996. 99–112.
Joyner A, Wall N. Immunohistochemistry of whole-mount mouse embryos. Cold Spring Harbor Protocols 2008; 2
Shadnia S, Soltaninejad K, Heydari K, Sasanian G, Abdollahi M. Tramadol intoxication: a review of 114 cases. Hum Exp Toxicol 2008; 27
Chevillard L, Declèves X, Baud F, Risède P, Mégarbane B. Respiratory effects of diazepam/methadone combination in rats: a study based on concentration/effect relationships. Drug Alcohol Depend 2013; 131
Nebhinani N, Singh S, Gupta G. A patient with tramadol dependence and predictable provoked epileptic seizures. Indian J Psychiatry 2013; 55
Bramness JG, Kornør H. Benzodiazepine prescription for patients in opioid maintenance treatment in Norway. Drug Alcohol Depend 2007; 90
Kaoud HA, Hellal MH, Malhat FM, Saeid S, Elmawella IA, Khalil AH. Effects of acute sub-lethal dose of tramadol on α2-adreergic receptors and liver histopathology in rat. Global J Curr Res 2013; 1
Cicero TJ, Inciardi JA, Adams EH, Geller A, Senay EC, Woody GE, et al.
Rates of abuse of tramadol remain unchanged with the introduction of new branded and generic products: results of an abuse monitoring system, 1994–2004. Pharmacoeoldemiol Drug Saf 2005; 14
Friedman A, Fadem S. Reassessment of albumin as a nutritional marker in kidney disease. JASN 2010; 21
Elyazj N, Abdel-Aziz I, Aldalou A, Shahwan O. The effects of tramadol hydrochloride administration on the hematological and biochemical profiles of domestic male rabbits. IUG J Nat Eng Stud 2013; 21
Bahman M, Reza A, Hossien N. Evaluation of prophylactic and therapeutic effects of silymarin on diazepam-induced hepatotoxicity in cat. Am J Appl Sci 2011; 8
Jeboori K, Ali A. Adverse effect of diazepam on cytogenetic and biochemical effects in white mice fed diet supplement with chitosan. GJBB 2014; 3
Musavi S, Kakkar P. Effect of diazepam treatment and its withdrawal on pro/antioxidative processes in rat brain. Mol Cell Biochem 2003; 245
Gonga L, Stamerc U, Tzvetkove M, Altmana R, Klein T, Pharm GKB. Summary: tramadol pathway. Pharmacogenet Genomics 2014; 24
Davanzo R, Dal BoS, Bua J, Copertino M, Zanelli E, Matarazzo L. Antiepileptic drugs and breastfeeding. Ital J Pediatr 2013; 39
Clarot F, Goulle JP, Vaz E, Proust B. Fatal overdoses of tramadol: is benzodiazepine a risk factor of lethality?. Forensic Sci Int 2003; 134
Tien LT, Ma T, Fan LW, Loh HH, Ho IK. Autoradiographic analysis of GABAA receptors in mu-opioid receptor knockout mice. Neurochem Res 2007; 32
Poisnel G, Dhilly M, Le Boisselier R, Barre L, Debruyne D. Comparison of five benzodiazepine-receptor agonists on buprenorphine-induced mu-opioid receptor regulation. J Pharmacol Sci 2009; 110
Mohamed TM, Ghaffar HM, El Husseiny RM. Effects of tramadol, clonazepam, and their combination on brain mitochondrial complexes. Toxicol Ind Health 2015; 31
Yin D, Woodruff M, Zhang Y, Whaley S, Miao J, Ferslew K, et al.
Morphine promotes Jurkat cell apoptosis through pro-apoptotic FADD/P53 and anti-apoptotic PI3K/Akt/NF-kappa B pathways. J Neuroimmunol 2006; 174
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]