Ameliorating Effect of a Beta-Blocker, Propranolol on Carbamazepine-Induced Hepatotoxicity in Rabbits

Hina Abrar1,2, S.N.H. Naqvi1,*, Muhammad Rashid Ahmed3, Asma Basharat Ali3 and Hina Yasin1,2

1Department of Pharmacology, Baqai Medical University, Super Highway, Gadap Road, P.O Box-2407, Karachi 74600

2Dow College of Pharmacy, Dow University of Health Sciences, Ojha Campus, Gulzar e Hijri, Scheme-33, Suparco Road, Karachi 75270

3Department of Anatomy, Baqai Medical University, Super Highway, Gadap Road, P.O Box-2407, Karachi 74600

ABSTRACT

Propranolol is non cardioselective beta blocker used to treat various cardiac and non-cardiac diseases including arrhythmia, hypertension, portal hypertension and oesophageal varices. The study was undertaken in rabbits to investigate the effect of propranolol to reduce hepatoxicity of carbamazepine (CBZ). Animals were divided into three groups; control, CBZ administered group (200 mg/Kg for 10 days) and CBZ plus propranolol (30 mg/Kg for 10 days) treated group. Liver function test and histological evaluation by H and E staining and scanning electron microscopy (SEM) was carried at the end of dosing by using standard procedures. Serum level of ALT, ALP, γGT and bilirubin was significantly (p<0.05) increased in CBZ treated group as compared to control, whereas the hepatic parameters were significantly reduced in CBZ plus propranolol group. The histopathological examination reveals various features of hepatic architecture damage in CBZ treated group but the hepatic damage induced by CBZ was successfully ameliorated by propranolol. To conclude propranolol is effective in reducing the hepatoxic effects of CBZ probably by affecting hepatic blood flow.

Article Information

Received 04 November 2016

Revised 13 April 2017

Accepted 31 May 2018

Available online 11 January 2019

Authors’ Contribution

HA, MRA, ABA and HY conceived and designed the study, collected the data and wrote the manuscript. SNHN supervised the research.

Key words

Hepatoxicity, Portal hypertension, Oesophageal varices carbamazepine, Propranolol.

DOI: http://dx.doi.org/10.17582/journal.pjz/2019.51.1.341.346

* Corresponding author: naeemnaqvi@live.com

0030-9923/2019/0001-0341 $ 9.00/0

Copyright 2019 Zoological Society of Pakistan

Introduction

Drug induced liver diseases are the commonest problem seen with majority of the pharmaceutical products. Drugs produce wide range of hepatic disorder ranging from asymptomatic jaundice to liver cirrhosis leading to life threatening hepatic failure (Chen et al., 2015). It is estimated that 1 in 100 patients have faced the problem of drug induced liver injury during hospitalization (Tarantino et al., 2009). In USA it is reported that 50% cases of hepatic failures are drug induced (Chalasani et al., 2008; Zheng and Navarro, 2016). The susceptibility of drug induced liver damage depends on several risk factors like age, sex, poly pharmacy, concomitant disease state and genetic polymorphism of metabolic pathways etc. (Chalasani and Bjornsson, 2010).

Different classes of drugs are well reported to cause hepatic injury including antimicrobial, antineoplastic, NSAIDs, antidiabetics, antitubercular drugs, anticonvulsants, antibiotics and antihyperlipidimics (Chalasani et al., 2015). Carbamazepine (CBZ) is one of the anticonvulsant drugs that is widely used to treat partial tonic clonic seizure in children and adults. Besides of its extensive use, many side effects has been reported including hepatotoxicity (George et al., 2016). CBZ related hepatotoxicity is due to metabolic and immunologic factors (Higuchi et al., 2012). Oxidative stress involvement is the basic mechanism of CBZ induced hepatotoxicity (Santos et al., 2008; Elliott et al., 2012). Arene oxide is the toxic metabolite of CBZ which is also reported to cause hepatotoxicity (Pandit et al., 2012). Numerous studies have been conducted to test various herbal formulation and vitamins to prevent the hepatotoxicity of CBZ (Santhrani et al., 2013; Maheswari et al., 2014; Shi et al., 2014).

Propranolol is effective in the prevention of esophageal variceal bleeding (Tursi, 2010). This effect of propranolol is due to reduction in portal blood flow (Bosch et al., 1984; Ohnishi et al., 1985; Pizcueta et al., 1989). As propranolol reduces the portal blood flow, it is our scientific belief that propranolol might be effective in reducing the hepatotoxicity of CBZ. Therefore, present study was designed to focus the effect of reduced hepatic blood flow induced by propranolol in reduction of hepatoxicity of CBZ.

 

Materials and Methods

Thirty six healthy male rabbit of weight 1.2 to 1.4 Kg were recruited from the animal house of Baqai Medical University, Karachi, Pakistan. All the animals were acclimatized for housing condition before starting the experiment. Each animal was kept in separate cage under controlled climatic condition during entire study in an alternating 12 h light and dark cycle. All animals had full access to water and standard laboratory food ad libitum.

All the animals were randomly divided into three groups, each comprised of 12 animals. Drugs were administered orally as follows: One group received distilled water orally for 10 days (control). The second group received CBZ 200mg/kg dissolved in distilled water orally for 10 days (Mesdjian et al., 1996). The third group received CBZ 200mg/kg and propranolol 30mg/kg orally for 10 days (Huang et al., 1998). After 24 h of last dose, the thoracic cage was exposed, approximately 5ml of blood was collected from each rabbit by cardiac puncture technique (Parasuraman et al., 2010). Blood sample were then transferred into gel tube and sent to the laboratory, where serum was separated by centrifugation at 4000rpm for 8 min. Alkaline phosphatase (ALP), alanine transaminase (ALT/SGPT) and γ-glutamyl transaminase (γ GT) and total bilirubin were estimated within 2 h of serum separation on automatic analyzer using standard kits purchased from Merck.

All the animals were sacrificed and livers were collected, flushed with saline in 10% normal buffered formalin for histopatholgical evaluation. After 24 h, liver tissues were embedded in paraffin wax as standard protocol. Five micrometer thick section were carried out from these block and put into poly-1-lysine coated glass slide and stained with haemotoxylin and eosin as standard procedure (Piao et al., 2016). The slides were observed under light microscope for histological changes induced by CBZ alone and in combination with propranolol. In micrometric studies number of intact hepatocytes, diameter of hepatocyte and diameter of nucleus were analyzed.

The formalin fixed tissues of liver were dehydrated by standard procedure. The samples were mounted on specimen stub using electrically conductive double sided adhesive tape and sputter coated with gold before examination in electron microscope (Echlin, 2011).

All the quantitative results were analyzed statistically using SPSS Software (Version 21). All the values were compared with control by taking mean and standard errors of mean (SEM) using ANOVA, considered p<0.05 was significant.

 

Results

Liver function

The serum analysis of bilirubin and liver enzymes SGPT (ALT), ALT, GGT between control and treated groups were used for assessment of hepatic injury. Statistical analysis showed the significant increase in all above parameters in CBZ treated group as compared to control and CBZ plus propranolol treated group (p<0.05). All these biochemical parameters were significantly reduced in CBZ plus propranolol treated group although, the values were more than the control.

Histological structure of liver

Control group specimens showed normal architecture of hepatic tissue with hepatocytes radiating from a central vein separated by normal sinusoidal spaces. The hepatocytes were polygonal with round nucleus with surrounding eosinophilic cytoplasm (Fig. 1A, B, C).

The morphological examination of the H & E stained liver sections of CBZ-treated animals showed distorted hepatic cord with marked mononuclear cell infiltrations in the periportal and pericentral area (Fig. 1D). The portal and central veins were also dilated and congested (Fig. 1E). Minimal swelling and marked congestions of sinusoids were also observed. At 400X the pyknotic necrosis of hepatocytes with cytoplasmic vacuolization were also noted. At 1000X inflammatory patches were observed and almost completely lost hepatic cell were seen. Hemorrhagic necrosis was also observed (Fig. 1F).

Liver sections of carbamazepine and propranolol treated liver showed preservation of hepatic architecture with minimal inflammatory cell infiltration. The central vein demonstrated mild dilation but the sinusoids appeared nearly normal. The pericentral area showed mild inflammatory cells infiltration. At 400X Slight mononuclear cell infiltrations were observed in portal tract with moderate portal vein dilation. At 1000X the polyhedral structure of hepatocytes were restored with normal nucleus and nucleolus (Fig. 1G, H, I).

The surface structure of section of liver of control rabbits showed normal hepatocyte cords and regular polyhyderal structure of hepatocyt (Fig. 2A). SEM of Carbamazepine treated liver sample showed marked changes in the surface of hepatocytes. There is multiple bleb formation in the cell membrane of the hepatocytes and swelling and rounding of hepatic cells are observed in group B (Fig. 2B). While SEM micrograph of CBZ plus propranolol treated group showed restoration of normal hepatocytes surface. The blebs have disappeared and polyhederal shape of hepatocytes was preserved (Fig. 2C).

 

Table I.- Effect of propranolol on liver of carbamazepine (CBZ) treated rabbits.

Parameters	Control (n=12)	CBZ treated (n=12)	CBZ + propranolol treated (n=12)
Liver function test
ALT(IU/L)	40.80 ±1.14	155.60±3.94*	118.40 ± 4.21**
ALP(IU/L)	42.60±2.79	71.20±4.01*	52.80±4.93**
γ GT (IU/L)	8.00 ± 0.93	17.00 ±1.50*	10.80 ±1.82**
Total bilirubin (m.mol/L)	0.50 ± 0.06	3.16 ± 0.39*	1.06 ± 0.12**
Micrometric parameters of liver
Hepatocyte count (cell/reticule)	21.70± 1.13	14.30± 0.91*	16.70±1.01**
Hepatocyte diameter (µm)	13.90± 0.33	18.70± 0.54*	15.18± 0.58**
Nuclear diameter (µm)	5.89± 0.07	4.46± 0.12*	5.24± 0.14**

Data are expressed as Mean+SEM (standard error of mean). *Significant when compare with control. **Significant when compare with CBZ treated group. ALT, alanine transaminase; ALP, alkaline phosphatase; γ GT, γ-glutamyl transaminase.

 

Number of hepatocytes, hepatocyte diameter and nuclear diameter were evaluated in all groups. The number of viable hepatocytes were significantly reduced in CBZ treated group when comparing with control. It was also reduced in CBZ plus propranolol treated group but not as significant as in CBZ treated group. CBZ significantly increased the hepatocytes diameter when comparing with control while propranolol reduced the CBZ induced cellular swelling. While the nuclear diameters were significantly reduced in CBZ treated group when comparing with control while this nuclear diameter reduction is insignificant (Table I).

 

Discussion

Antiepileptic drugs are widely used in the treatment of various psychiatric and neurological disorders. Therapeutic use of AEDs (aromatic anti epileptic drugs) is usually associated with the elevated liver enzymes. These are therapeutically monitored drugs because of high potential of toxicity (Ahmed and Siddiqi, 2006). Involvement of oxidative stress is the major mechanism of CBZ induced hepatotoxicity. Several antioxidant treatments have been reported to reduce the hepatotoxicity of CBZ (Maheswari et al., 2015a, b).

Propranolol is a beta blocking agent and binds to the G-protein coupled adrenoceptors, is an effective portal hypotensive agent (Lee et al., 2011). Propranolol is commonly used to treat portal hypertension, cirrhosis and oesophageal varices. It is also effective in reducing bacterial translocation which is suggested to prevent hepatocellular carcinoma and in prevention of hepatocellular carcinoma followed by liver cirrhosis (Pérez-Paramo et al., 2000). Beside reduction of hepatic blood flow propranolol and its metabolite are accountable as strong antioxidant agent (Adali et al., 1999; Mak and Weglicki, 2004). The present study was specially designed to investigate the extent of CBZ toxicity on liver and reduction of the hepatotoxic features of CBZ with the help of propranolol.

Liver function test is the best way to evaluate the status of liver (Kim and Younossi, 2008; Thapa and Walia, 2007). In this study, the levels of serum ALT (SGPT), ALP, and GGT enzymes and bilirubin were measured which are commonly used hepatobiliary biomarkers. Increased SGPT (ALT) activity indicates hepatocellular injury while increased ALP and GGT activities showed obstruction in the bile flow (Cengiz et al., 2017). The present study revealed that the levels of ALT, ALP, γGT and bilirubin were considerably increased in CBZ treated group as compared to control and CBZ plus propranolol treated group. The results of biochemical estimation showed that CBZ induced liver injury was indicated by increased hepatic biomarker. These hepatic biomarkers were remarkably reduced by co-administration of propranolol.

Histological examination of liver tissues showed CBZ cause mononuclear cell infiltration in portal tract along with sinusoidal dilatation and central vein congestion, these observations are robustly correlated with the results given by other researchers (Santhrani et al., 2013; Maheswari et al., 2015a). On the other hand the quantitative microscopic evaluation illustrated that there is reduction in the viable hepatocyte count along with swelling of hepatocytes indicated by increased diameter of hepatocytes. Nuclear diameter was also reduced in CBZ treated group which indicates the reduction in cell viability. The ballooning degeneration of hepatocytes and decreased activity of cells after CBZ administration were also previously reported (Sasaki et al., 2016; Eghbal et al., 2013). When compared these interpretations of histological examination with CBZ and propranolol treated group it was demonstrated that the combination of propranolol reversed the hepatic damage induced by CBZ. The hepatic architecture was effectively restored when propranolol was co-administered with CBZ.

Thus all the above findings suggested that the hepatotoxicity induced by CBZ was noticeably reduced by concurrent administration of propranolol. Propranolol reduced and reversed the hepatotoxicity and portal hypertension induced by ethanol (Prkacin et al., 2001). This effect of propranolol is due to reduced hepatic blood flow by portal vascular contraction. The diameter of central vein was also reduced by propranolol. It was well understood that in heptotoxicity the blood flow of liver was increased as adaptive response to combat hypoxia. Propranolol furthermore possesses an antioxidant effect which was also helpful in reducing the toxicity of CBZ.

 

Conclusion

The present study was carried out to investigate the role of propranolol on various hepatic parameters. Propranolol was co administered with CBZ for specified period of time the results showed that CBZ induced significant hepatoxicity and these observations were confirmed by qualitative and quantitative microscopic examinations and scanning electron microscopy. Propranolol profoundly reduced the hepatotoxicity observed in biochemical and microscopic evaluations. This effect of propranolol is due to reduced hepatic blood flow. However, further advanced studies are required to confirm these results.

 

Statement of conflict of interest

Authors have declared no conflict of interest.

 

References

Adali, M., İnal-Erden, Ṁ., Akalin, A. and Efe, B.N., 1999. Effects of propylthiouracil, propranolol, and vitamin E on lipid peroxidation and antioxidant status in hyperthyroid patients. Clin. Biochem., 32: 363-367. https://doi.org/10.1016/S0009-9120(99)00024-7

Ahmed, S.N. and Siddiqi, Z.A., 2006. Antiepileptic drugs and liver disease. Seizure, 15: 156-164. https://doi.org/10.1016/j.seizure.2005.12.009

Bosch, J., Masti, R., Kravetz, D., Bruix, J., Gaya, J., Rigau, J. and Rodes, J., 1984. Effects of propranolol on azygos venous blood flow and hepatic and systemic hemodynamics in cirrhosis. Hepatology, 4: 1200-1205. https://doi.org/10.1002/hep.1840040617

Cengiz, M., Ali, J.H., Kutlu, M,, Vejselova, D. and Ayhanci, A., 2017. Potential recruiting and hepatoprotective effects of ellagic acid in D-galactosamine-induced liver damage in rats. Pakistan J. Zool., 49: 1251-1259

Chalasani, N. and Bjornsson, E., 2010. Risk factors for idiosyncratic drug-induced liver injury. Gastroenterology, 138: 2246-2259. https://doi.org/10.1053/j.gastro.2010.04.001

Chalasani, N., Bonkovsky, H.L., Fontana, R., Lee, W., Stolz, A., Talwalkar, J., Reddy, K.R., Watkins, P.B., Navarro, V., Barnhart, H., Gu, J. and Serrano, J., 2015. Features and outcomes of 899 patients with drug-induced liver injury: The DILIN prospective study. Gastroenterology, 148: 1340-1352. https://doi.org/10.1053/j.gastro.2015.03.006

Chalasani, N., Fontana, R.J., Bonkovsky, H.L., Watkins, P.B., Davern, T., Serrano, J., Yang, H. and Rochon, J., 2008. Causes, clinical features, and outcomes from a prospective study of drug-induced liver injury in the United States. Gastroenterology, 135: 1924-1934. https://doi.org/10.1053/j.gastro.2008.09.011

Chen, M., Suzuki, A., Borlak, J., Andrade, R.J. and Lucena, M.I., 2015. Drug-induced liver injury: Interactions between drug properties and host factors. J. Hepatol., 63: 503-514. https://doi.org/10.1016/j.jhep.2015.04.016

Echlin, P., 2011. Handbook of sample preparation for scanning electron microscopy and X-ray microanalysis. Springer Science & Business Media.

Eghbal, M.A., Taziki, S. and Sattari, M.R., 2013. Protective role of melatonin and taurine against carbamazepine-induced toxicity in freshly isolated rat hepatocytes. Int. J. Morphol., 1: 1081-1089. https://doi.org/10.4067/S0717-95022013000300049

Elliott, E.C., Regan, S.L., Maggs, J.L., Bowkett, E.R., Parry, L.J., Williams, D.P., Park, B.K. and Stachulski, A.V., 2012. Haloarene derivatives of carbamazepine with reduced bioactivation liabilities: 2-monohalo and 2,8-dihalo derivatives. J. med. Chem., 55: 9773-9784. https://doi.org/10.1021/jm301013n

George, M., Joseph, L. and Jose, P.C., 2016. A review article on assessing the effect of antiepileptics and statins on liver enzymes in epileptic patients. Pharma Innov. J., 5: 11-14.

Higuchi, S., Yano, A., Takai, S., Tsuneyama, K., Fukami, T., Nakajima, M. and Yokoi, T., 2012. Metabolic activation and inflammation reactions involved in carbamazepine-induced liver injury. Toxicol. Sci., 130: 4-16. https://doi.org/10.1093/toxsci/kfs222

Huang, Y.T., Cheng, Y.R., Lin, H.C., Hou, M.C., Lee, S.D. and Hong, C.Y., 1998. Hemodynamic effects of eight-day octreotide and propranolol administration in portal hypertensive rats. Digest. Dis. Sci., 43: 358-364. https://doi.org/10.1023/A:1018866608377

Kim, C.H. and Younossi, Z.M., 2008. Nonalcoholic fatty liver disease: A manifestation of the metabolic syndrome. Cleveland Clin. J. Med., 75: 721-728. https://doi.org/10.3949/ccjm.75.10.721

Lee, J.Y., Huo, T.I., Huang, H.C., Lee, F.Y., Lin, H.C., Chuang, C.L., Chang, C.C., Wang, S.S. and Lee, S.D., 2011. Propranolol modulates the collateral vascular responsiveness to vasopressin via a Gα-mediated pathway in portal hypertensive rats. Clin. Sci., 121: 545-554. https://doi.org/10.1042/CS20100590

Maheswari, E., Saraswathy, G.R. and Santhranii, T., 2014. Hepatoprotective and antioxidant activity of N-acetyl cysteine in carbamazepine-administered rats. Indian J. Pharmacol., 46: 211-215. https://doi.org/10.4103/0253-7613.129321

Maheswari, E., Saraswathy, G.R., Raja, G. and Santhranii, T., 2015a. Effectiveness of alpha lipoic acid as hepatoprotective and antioxidant. Int. J. Pharmaceut. Chem. biol. Sci., 5: 242-248.

Maheswari, E., Saraswathy, G. and Santhranii, T. 2015b. Influence of vitamin E on hepatotoxicity and oxidative stress. Int. J. Res. Pharm. Biosci., 2: 30-38.

Mak, I.T. and Weglicki, W.B., 2004. Potent antioxidant properties of 4-hydroxyl-propranolol. J. Pharmacol. exp. Therap., 308: 85-90.

Mesdjian, E., Zamora, A.J., Montet, A.M., Bonneton, J., Guitaoui, M., Genton, P. and Montet, J. C. 1996. Ursodeoxycholate improves hepatobiliary dysfunction induced by valproate-carbamazepine treatment in the rat. Life Sci., 59: 1069-1079. https://doi.org/10.1016/0024-3205(96)00422-5

Ohnishi, K., Nakayama, T., Saito, M., Hatano, H., Tsukamoto, T., Terabayashi, H., Sugita, S., Wada, K., Nomura, F., Koen, H. and Okuda, K., 1985. Effects of propranolol on portal hemodynamics in patients with chronic liver disease. Am. J. Gastroenterol., 80: 132-135.

Pandit, A., Sachdeva, T. and Bafna, P., 2012. Drug-induced hepatotoxicity: A review. J. appl. Pharmaceut. Sci., 2: 233-243. https://doi.org/10.7324/JAPS.2012.2541

Parasuraman, S., Raveendran, R. and Kesavan, R., 2010. Blood sample collection in small laboratory animals. J. Pharmacol. Pharmacother., 1: 87–93. https://doi.org/10.4103/0976-500X.72350

Pérez-Paramo, M., Munoz, J., Albillos, A., Freile, I., Portero, F., Santos, M. and Ortiz-Berrocal, J., 2000. Effect of propranolol on the factors promoting bacterial translocation in cirrhotic rats with ascites. Hepatology, 31: 43-48. https://doi.org/10.1002/hep.510310109

Piao, M., Liu, Y., Yu, T. and Lu, Y., 2016. Zinc supplementation ameliorates ER stress and autophagy in liver in a rat model of type 2 diabetes mellitus. Biomed. Res., 27: 970-938.

Pizcueta, M.P., de Lacy, A.M., Kravetz, D., Bosch, J. and Rodés, J., 1989. Propranolol decreases portal pressure without changing portocollateral resistance in cirrhotic rats. Hepatology, 10: 953-957. https://doi.org/10.1002/hep.1840100610

Prkacin, I., Separovic, J., Aralicia, G., Perovic, D., Gjurasin, M., Lovric-Bencic, M., Stancic-Rokotov, D., Staresinic, M., Anic, T. and Mikus, D., 2001. Portal hypertension and liver lesions in chronically alcohol drinking rats prevented and reversed by stable gastric pentadecapeptide BPC 157 (PL-10, PLD-116), and propranolol, but not ranitidine. J. Physiol., 95: 315-324. https://doi.org/10.1016/S0928-4257(01)00044-4

Santhrani, T., Maheswari, E. and Saraswathy, G.R., 2013. Carbamazepine provoked hepatotoxicity: Attenuation by vitamin C. Oxidant. Antioxi. med. Sci., 2: 37-43.

Santos, N.A., Medina, W.S., Martins, N.M., Rodrigues, M.A., Curti, C. and Santos, A.C., 2008. Involvement of oxidative stress in the hepatotoxicity induced by aromatic antiepileptic drugs. Toxicol. In Vitro, 22: 1820-1824. https://doi.org/10.1016/j.tiv.2008.08.004

Sasaki, E., Iida, A., Oda, S., Tsuneyama, K., Fukami, T., Nakajima, M. and Yokoi, T., 2016. Pathogenetic analyses of carbamazepine-induced liver injury in F344 rats focused on immune-and inflammation-related factors. Exp. Toxicol. Pathol., 68: 27-38. https://doi.org/10.1016/j.etp.2015.09.004

Shi, L., Dang, X.L., Liu, X.Y., Wei, H.M., Yang, M.M. and Zhang, Y., 2014. Effect of Sophora flavescens on the pharmacokinetics of carbamazepine in rats. Arch. Pharm. Res., 37: 1617-1623. https://doi.org/10.1007/s12272-014-0375-8

Tarantino, G., Di Minno, M.N. and Capone, D., 2009. Drug-induced liver injury: Is it somehow foreseeable? World J. Gastroenterol., 15: 2817-2833. https://doi.org/10.3748/wjg.15.2817

Thapa, B. and Walia, A., 2007. Liver function tests and their interpretation. Indian J. Pediat., 74: 663-671. https://doi.org/10.1007/s12098-007-0118-7

Tursi, T., 2010. Use of ss-blocker therapy to prevent primary bleeding of esophageal varices. J. Am. Acad. Nurse Pract., 22: 640-647. https://doi.org/10.1111/j.1745-7599.2010.00567.x

Zheng, E. and Navarro, V., 2016. Drug-induced liver injury in the United States: A review of multi-ingredient supplements. Clin. Liver Dis., 7: 60-63. https://doi.org/10.1002/cld.535