|Year : 2018 | Volume
| Issue : 1 | Page : 40-47
Impact of benzodiazepines administration on selected biochemical parameters of albino Wistar rats (Rattus rattus)
Joyce N Nzor, Augustin A Uwakwe, Samuel C Onuoha
Department of Biochemistry, University of Port Harcourt, Port Harcourt, Rivers, Nigeria
|Date of Web Publication||4-May-2018|
Joyce N Nzor
Department of Biochemistry, Faculty of Science, University of Port Harcourt, Port Harcourt, Rivers, 500102
Source of Support: None, Conflict of Interest: None
Background Considerable information has been reported on the adverse effect of benzodiazepines on the brain, with limited information of their effects on biochemical parameters.
Aim This study aimed to investigate the effect of diazepam and bromazepam on selected biochemical parameters of albino Wistar rats.
Materials and methods Diazepam and bromazepam at concentrations of 0.0046 mg/100 g body weight, 0.0036 mg/100 g body weight, 0.0026 mg/100 g body weight, and 0.0016 mg/100 g body weight were administered, respectively, to rats on a daily basis. At the end of each week, total serum protein, glucose, urea, and creatinine levels were evaluated. Histopathological examination of the liver was also performed for signs of possible damage.
Results Reductions in serum glucose and creatinine levels were observed in the first, second, third, and fourth weeks in rats administered diazepam and bromazepam at various doses. Both drugs showed similar effects and the reductions were only significant (P<0.05) for creatinine, whereas total serum protein was elevated significantly (P<0.05). No effect was observed in urea levels throughout the study period. Histopathological examination of the liver showed pronounced morphological alterations in structure, indicative of hepatic damage.
Conclusion Although hematological parameters such as glucose and urea were not affected significantly, the observed hepatotoxicity could be indicative of possible induction of hepatic damage with consequent metabolic aberrations.
Keywords: bromazepam, creatinine, diazepam, glucose, hepatocytes, protein, urea
|How to cite this article:|
Nzor JN, Uwakwe AA, Onuoha SC. Impact of benzodiazepines administration on selected biochemical parameters of albino Wistar rats (Rattus rattus). Egypt Pharmaceut J 2018;17:40-7
|How to cite this URL:|
Nzor JN, Uwakwe AA, Onuoha SC. Impact of benzodiazepines administration on selected biochemical parameters of albino Wistar rats (Rattus rattus). Egypt Pharmaceut J [serial online] 2018 [cited 2018 Nov 20];17:40-7. Available from: http://www.epj.eg.net/text.asp?2018/17/1/40/231876
| Introduction|| |
Anxiolytics are prescribed worldwide for the relief of pain, of which benzodiazepines are among the most frequently prescribed ,. Benzodiazepines were introduced in the 1950s and are minor tranquilizers that act as central nervous system depressants through the facilitation of γ-aminobutyric acid binding at various γ-aminobutyric acid receptors throughout the central nervous system . Hence, benzodiazepines are effective for the following therapeutic actions in short-term usage: anxiolytic (anxiety and panic disorder), myorelaxant (muscle spasms, spastic disorders), anticonvulsant (some forms of epilepsy, fits because of drug poisoning), hypnotic (insomnia), and amnesia (sedation for minor operations) . Both diazepam and bromazepam belong to the 1,4-benzodiazepin-2-one class of benzodiazepines  and considerable information has been reported on the adverse effects of benzodiazepines on the brain ,, with limited information of its effect on biochemical parameters.
This study aimed to investigate the effects of diazepam and bromazepam on selected biochemical parameters of albino Wistar rats; the aim was to observe possible changes in serum total blood protein, serum glucose, serum creatinine, and serum urea, and also perform histological examination of the liver.
| Materials and methods|| |
Approval was obtained from the ethics committee of University of Port Harcourt for the use of animals for experiments, after which 108 albino Wistar rats weighing between 100 and 110 g of both sexes were obtained from the animal house of the University of Port Harcourt, Choba, Rivers State, Nigeria; acclimatization was allowed for 1 week before experimentation. Water and standard rat chow were provided ad libitum and the animals were kept under room temperature. At the end of each week, three rats were killed from each group and the animals were divided into nine groups as follows: group 1 (DZ1), which consisted of 12 rats administered 0.0046 mg/100 g body weight of diazepam orally, group 2 (DZ2), which consisted of 12 rats administered 0.0036 mg/100 g body weight of diazepam orally, group 3 (DZ3), which consisted of 12 rats administered 0.0026 mg/100 g body weight of diazepam orally, group 4 (DZ4), which consisted of 12 rats administered 0.0016 mg/100 g body weight of diazepam orally, group 5 (BZ1), which consisted of 12 rats administered 0.0046 mg/100 g body weight of bromazepam orally, group 6 (BZ2), which consisted of 12 rats administered 0.0036 mg/100 g body weight of bromazepam orally, group 7 (BZ3), which consisted of 12 rats administered 0.0026 mg/100 g body weight of bromazepam orally, group 8 (BZ4), which consisted of 12 rats administered 0.0016 mg/100 g body weight of bromazepam orally, and group 9 (control group), which consisted of 12 untreated normal rats.
Drugs and chemicals
Bromazepam (lexotan) tablet and diazepam (valium) used were manufacturred by F. Hoffmann-La Roche Ltd (Basel, Switzerland), the randox kit used was obtained from Randox chemical Laboratory (Crumlin, UK), and all other reagents used were of analytical grade.
Collection of blood and organ
The animals were killed under anesthesia (chloroform suffocation) and blood samples were collected into plain sample bottles. The liver was stored in plain bottles containing 75% formalin before embedding in parafin wax and further processed for examination . Serum was separated at 3000 rpm for 10 min and stored in a refrigerator at −4°C for subsequent analysis.
Estimation of biochemical parameters
Serum creatinine was determined using the randox kit (Jaffe’s reaction method) . The glucose level was determined using the randox test kit (glucose oxidase PAP method) , whereas the serum urea level was determined using the diacetyl monoxime method and serum total protein was estimated using the biuret reagent method.
Analysis of results
Data obtained from the experimental design and set-up were subjected to statistical calculations using SPSS, version 18 (SPSS Inc., Chicago, IL, USA). The mean values±SD were calculated and the one-way analysis of variance test was performed. Significance level was calculated at a 95% confidence level (P≤0.05).
| Results|| |
The results obtained from the various analyses of the effect of diazepam and bromazepam of some biochemical parameters are presented in [Table 1],[Table 2],[Table 3],[Table 4]. Nonsignificant reductiouns in serum glucose concentrations were observed at varied concentrations of the two drugs, whereas concentration-dependent increases in serum protein concentrations were observed in the presence of the drugs. No significant effect on the serum urea concentration was observed in the presence of the drugs at the concentrations investigated.
|Table 1 Effect of diazepam and bromazepam on serum glucose concentration|
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|Table 2 Effects of diazepam and bromazepam on serum protein concentration|
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|Table 3 Effects of diazepam and bromazepam on serum urea nitrogen concentration|
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|Table 4 Effects of diazepam and bromazepam on serum creatinine concentration|
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Histopathological examination of the liver indicated signs of inflammation, congested vessels, dilated sinusoids, and vacuolar degeneration. Damage to the liver was observed only at the third and fourth week, with higher doses causing more damage. Histopathology of the liver at the third and fourth week is shown in Plates 1-17
| Discussion|| |
Effect of diazepam and bromazepam on serum glucose concentration
Data from this study indicated that the administration of diazepam (0.0046 mg/100 g body weight, 0.0036 mg/100 g body weight, 0.0026 mg/100 g body weight, and 0.0016 mg/100 g body weight), respectively, and bromazepam (0.0046 mg/100 g body weight, 0.0036 mg/100 g body weight, 0.0026 mg/100 g body weight, and 0.0016 mg/100 g body weight) produced a nonsignificant reduction in the serum glucose concentration throughout the duration of the experiment ([Table 1]). However, 0.0046 mg/100 g body weight of the drugs caused a significant reduction (P<0.05) compared with the control as an initial effect.
In this study, it was found that diazepam and bromazepam had no effect on serum glucose levels. This finding is in agreement with the previous research work ,, whose results showed no significant reduction in blood glucose levels of patients and rabbits when diazepam was administered; the current clinical study found decreases in glucose levels of benzodiazepine patients, although the reduction was significant . On the basis of these results, it is clear that diazepam and bromazepam can be administered safely for the short-term treatment of anxiety or as a sedative for both diabetic and nondiabetic patients without the fear of alteration of blood glucose at these concentrations of diazepam and bromazepam.
Short-term administration of benzodiazepines leads to reduction or suppression of cortisol and adrenalin production through the inhibition of the corticotrophin-releasing factor in the hypothalamus–pituitary axis . However, with long-term administration, the receptors are no longer sensitive to the same concentration of benzodiazepine that initially produced that effect; thus, the cortisol and adrenalin levels may then increase.
Effect of diazepam and bromazepam on serum total protein concentration
Total serum protein increased significantly (P≤0.05) in a dose-dependent manner compared with the control; higher doses of both drugs led to higher concentrations of serum total proteins ([Table 2]). Subsequent administration showed a similar pattern, with no significant difference between both drugs at similar concentrations; bromazepam led to a greater increase in protein level than diazepam.
In a relaxed state/condition, the net synthesis of protein occurs as catabolic activities are reduced, protein synthesis has been linked to rapid eye movement sleep , and benzodiazepine serves as a hypnotic/sedative drug. Sleep recordings have shown that because of tolerance, the efficacy of the hypnotic effect declines, resulting in patterns of sleep suppressed originally by benzodiazepine returning to the same state before treatment started .
Effect of diazepam and bromazepam on kidney function markers (serum creatinine and serum urea)
The use of blood urea nitrogen as a marker for renal damage by diazepam and bromazepam showed no effect; the alteration in serum urea nitrogen throughout the duration of the experiment was minimal as shown in [Table 3]. However, an alteration in the serum creatinine concentration was caused by the drugs; diazepam and bromazepam significantly reduced the serum creatinine level in a dose-dependent manner (P<0.05) ([Table 4]).
Urea concentration was minimally affected by diazepam and bromazepam, indicating proper functioning of the kidney, but the creatinine concentration was reduced significantly. This condition (reduction in the creatinine concentration) only occurs when there is muscle wasting or overhydration and this was not the reason for the reduction in the creatinine concentration in this study as there was no weight loss in the experimental animals. However, the reduction in the creatinine concentration could be a result of a reduced metabolic rate leading to creatinine generation or a consequence of possibly increased renal clearance of this metabolite as suggested earlier by Tawfiq and colleagues ,.
Effect of diazepam and bromazepam on liver histology
Benzodiazepine liver injury has been classified as an unpredictable or an idiosyncratic hepatotoxic reaction , but this study showed that hepatotoxicity of benzodiazepine can also be studied in experimental animals. The liver indicated signs of inflammation, supporting the possibility of hepatotoxicity as reported in cholestasis liver injury cases ,.
| Conclusion|| |
The findings from this study suggest that diazepam and bromazepam may exert deleterious effects on the hepatocytes, with possible metabolic implications in the long run. Further research is required to confirm whether what was observed in the experimental animals can be replicated in humans. This would be necessary to establish the safety or otherwise of the administration of these drugs to those with an established history of hepatic damage or dysfunction. Moreover, studies on the possible effect of long-time administration of these drugs are also needed to fully establish their safety or otherwise in key metabolic systems.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Huxtable CA, Roberts LJ, Somogyi AA, Macintyre PE. Acute pain management in opioid-tolerant patients: a growing challenge. Anaesth Intensive Care 2011; 39:804–823.
Dixit RK, Puri JN, Sharma MK, Jain IP, Singh S, Ansari NA et al.
Effect of anxiolytics on blood sugar level in rabbits. Indian J Exp Biol 2001; 39:378–380.
Ashton H. The diagnosis and management of benzodiazepine dependence. Curr Opin Psychiatry 2005; 18:249–255.
Olfson M, King M, Schoenbaum M. Benzodiazepine use in the United States. JAMA Psychiatry 2015; 72:136–142.
Honeychurch KC, Hart JP. Electrochemical detection of benzodiazepines, following liquid chromatography, for applications in pharmaceutical, biomedical and forensic investigations. Insciences J 2014; 4:1–18.
Hicks D. Recreational drugs and their impact on diabetes. J Diabetes Nurs 2016; 20:110–114.
Kiernan JA. Histological and histochemical methods − theory and practice. 5th ed. Banbury, UK: Scion Publishing Ltd; 2015.
Syal K, Banerjee D, Srinivasan A. Creatinine estimation and interference. Indian J Clin Biochem 2013; 28:210–211.
Harish JP, Srinivas HA, Soumya AP. Comparative study of glucometer and laboratory glucose oxidase method for the estimation of blood glucose levels in neonates. J Evol Med Dent Sci 2015; 4:2652–2662.
Schaira VRL, Ranali J, Saad MJA, de Oliveira PC, Ambrosano GMB, Volpato MC. Influence of diazepam on blood glucose levels in nondiabetic and non-insulin-dependent diabetic subjects under dental treatment with local anesthesia. Anesth Prog 2004; 51:14–18.
Sanli DB, Bilici R, Suner O, Citak S, Kartkaya K, Mutlu FS. Effect of different psychoactive substances on serum biochemical parameters. Int J High Risk Behav Addict 2015; 4:e22702.
Gronli J, Soule J, Bramham CR. Sleep and protein synthesis-dependent synaptic plasticity: impacts of sleep loss and stress. Front Behav Neurosci 2013; 7:224.
Tawfiq RA, Nassar NN, El-Eraky WI, El-Denshary ES. Enhanced efficacy and reduced side effects of diazepam by kava combination. J Adv Res 2014; 5:587–594.
Andrade RJ, Lucena MI, Aguilar J, Lazo MD, Camargo R, Moreno P et al.
Chronic liver injury related to use of bentazepam: an unusual instance of benzodiazepine hepatotoxicity. Dig Dis Sci 2000; 45:1400–1404.
Larrey D, Ripault MP. Hepatotoxicity of psychotropic drugs and drugs of abuse. In: Kaplowitz N, deLeve LD, editors. Drug-induced liver disease. 3rd ed. Amsterdam, The Netherlands: Academic Press; 2013. p. 455.
[Table 1], [Table 2], [Table 3], [Table 4]