Effect of Haloperidol on Development of Hepatocytes in the Albino Rats

Introduction: Haloperidol is first generation antipsychotic used to treat psychosis. Its usage during pregnancy benefits psychotic mother and is indispensable for treating psychiatric emergency situations. Aims & Objectives: To evaluate the effects of haloperidol on development of liver given to albino rats in intrauterine period. Place and duration of study: Animal house PGMI, Bird wood Road. Duration of study is 2 months. Material & Methods: 45 female albino rats and 12 male rats of Sprague-dawley strain were used for conception. After conception, female rats were randomly divided into three groups A, B and C. Each group having 15 rats. Group A did not receive any drug whereas group B & C were given intraperitoneally haloperidol in a dose of 0.4mg/kg and 0.8mg/kg respectively. Hysterotomy was done on 21st day of gestation and pups were removed. Average litter size of pups in each group was 5-10 pups per female albino rat. These pups were labeled as A1, B1 and C1. They were grossly examined for any abnormality and liver was removed after dissection. Slides were made and stained to evaluate changes in detailed histological study of hepatocytes. Results: Comparison of diameter of hepatocytes in group B1 and C1 were significant from group A1 with p-value ≤0.001. However comparison of hepatic nucleus diameter among three groups were statistically insignificant with p-value 0.357. Conclusion: Haloperidol was responsible for destroying the normal architecture of the developing liver. Liver growth was shown by poor development of hepatocytes at high doses.


INTRODUCTION
Psychosis is a disease of neuronal disconnection caused by multiple genetic and environmental factors that affect brain development 1 . The genetic etiology is likely to be polygenic. Approximately 6.6 percent of all first degree relatives of an affected individual can be affected by psychosis 2 . Psychosis treatment involves education, medication, close monitoring of symptoms, stress management and creating a strong, supportive environment. Medication is important in relieving symptoms of psychosis and is critical in preventing relapses. They are called antipsychotics or neuroleptics 3 . Haloperidol was approved by the United States Food and Drug Administration (FDA) on April 12, 1967 4 ; it was later marketed in the U.S. and other countries under the brand name Haldol by McNeil Laboratories. Haloperidol is a first-generation (typical) neuroleptic, non-selective and binds to a broad range of receptors as dopamine D1 and D2, 5-HT2, histamine H1 and α2 adrenergic receptors in the brain. Its mechanism of action is due to antagonism of dopamine receptors in the mesolimbic and mesofrontal systems 5 . The monitoring of haloperidol is important clinically, however large doses can be given safely in intravenous and intramuscular injections for rapid neuroleptization 6 . Plasma haloperidol concentration varies and its hydroxyl metabolites are responsible for biological activity and prolonged clinical effects. The recommended dose in psychosis is 0.5 to 5 mg twice or thrice, intramuscular doses vary from 2 to 30 mg whereas intravenous up to 30mg 7 . Lethal dose (LD50) in rats is ≥128mg/kg subcutaneously, 27mg/kg intraperitoneally and 15mg/kg intravenously 8 . Haloperidol is contraindicated in coma, acute stroke cardiac disease, pregnancy and lactating mother. Various studies were conducted in the past to evaluate effects of haloperidol in different organs of adults and fetus. In a series of paper by Lewis et al, he concluded that haloperidol stunted brain growth 9 . In 1990, it was suggested that forebrain development was affected 10 with its usage and proliferation of brain cells were affected during development 11 . In 2002, increase no of apoptotic cells were seen in cerebral cortex 12 and vasoconstriction of basilar artery was observed in 2004 with chronic haloperidol usage 13 . Damaging effects on adult liver was observed in a study conducted in 2009 14 . In 2010, another study conducted on guinea pigs also revealed damaging effects of haloperidol on adult liver 15 . This study was designed to see the damaging effects of haloperidol on developing liver.

MATERIAL AND METHODS
45 female albino rats and 12 male albino rats of Sprague-drawly strain, weighing about 250-300 g were used. They were obtained from Pakistan Council of Scientific and Industrial Research (PCSIR), Karachi. All animals male and females were kept separately in the animal house of the Punjab Postgraduate Medical Institute, Lahore and were acclimatized for 15 days. A twelve hour light and dark cycle was maintained at room temperature between 22-25 0 C. After acclimatization three female and one male rats were kept together in a cage for a week for conception and male rats were removed from the cage later on. Female rats were observed for vaginal plug (Appendix II) 16 . This was taken as day zero of pregnancy. After conception female rats were randomly divided in three groups; A (control), B (experimental low dose) and C (experimental high dose), each group having 15 rats. Haloperidol in injectable form was given to the rats by intraperitoneal route (Fig-2) from 9 th day of gestation onwards as liver primordium first appears on the 11 th day of development 17 . The dose schedule was as follows: Control Group A: It had 15 female rats which were given 0.2mg/kg body weight of phosphate-buffered saline intra peritoneal from 9 th to 21 st day of gestation as a champ treatment. Experimental Group B: It had 15 female rats which were given 0.4mg/kg body weight of haloperidol intra peritoneal from 9 th to 21 st day of gestation. Experimental Group C: It had 15 female rats which were given 0.8mg/kg body weight of haloperidol intra peritoneal from 9 th to 21 st day of gestation. These animals of each group (A,B and C) were then euthanized on 21 st day of gestation by injecting sodium pentobarbital as anesthetic intraperitoneally in doses of 45mg/kg 18,19 and morphine as analgesic in doses of 0.3-0.5mg/kg intraperitoneally 20 . Hysterotomy was done and pups were removed. Average litter size of albino rats in each group was 5 per animal, so in this step of study the total number of the pups was 5 x 45 = 225. 24 pups from each group were randomly selected by lottery method (appendix-III) 21 . These pups were labeled as A1, B1, and C1.

Procedure of dissection:
The body weight of each animal was recorded before dissection, stretched out in supine position on the dissection tray. The limbs were fixed with the help of pins and a midline incision was given through the skin that extended from the xiphisternum to the pubic symphysis and abdominal wall was opened in the mid line with the help of scissors. The liver was identified, falciform and coronary ligaments were cut, common hepatic duct and hepatic vessels were incised and liver was dissected out. The liver was weighed and observed for any gross abnormality and preserved in 10% formalin for histological evaluation.

Measurement of size of Hepatocytes:
Under light microscopy, micrometry would be performed. In each slide 20 hepatocytes were selected randomly, size of each hepatocyte was measured with microscopic oculometer in transverse, anteroposterior and oblique dimensions. Average of these three dimensions was calculated to get average size of a hepatocyte. Then average of it was calculated. This average value was taken as final value.

Statistical analysis:
Data was analysed by SPSS version 21. Diameter of hepatocytes was described by Mean, ±S.D. and comparison between the groups were made by ANOVA. The P-value less than 0.05 was considered as statistically significant.

RESULTS
The mean diameter of hepatocyte in control group was 13.85±0.71 μm, while in group B1 it was recorded 12.07±0.95 μm and 12.04±1.45 μm in C1. The difference among three groups was statistically significant with p-value <0.001 (Table-1) Pair wise comparison yielded that the difference of groups B1 and C1 from A1 was both significant with p-value <0.001 but the difference between group B1 and C1 was statistically insignificant with p-value 0.996. (Table-2, Fig-1

DISCUSSION
In the present study the diameter of the hepatocytes and diameter of nucleus of the hepatocytes in experimental groups B1 and C1 were noted and compared with the control group A1. The results of this study revealed significantly decreased hepatocyte diameter in experimental groups B1 & C1 as compared with the control group A1 (p-value 0.001). Hepatic nuclear diameter, however seems to be unaffected in all the groups. The probable mechanisms for these reduced diameters are toxicity of haloperidol metabolites to liver cells resulting in cell atrophy and sequelae of cell adaptation to cell injury 22 . Injurious effects of haloperidol causes decreased synthesis of cellular binding blocks and increased breakdown of cellular organelles leading to atrophy of the cell. This may be due to the fact that haloperidol produces pyridine metabolites by cytochrome P450 mechanism which reduces glutathione inside the mitochondria and releases intracellular enzymes aminotransferases which are hepatic cell membrane injury markers 23 . Depletion of glutathione results in oxidative stress with depletion of ATP and loss of oxidative phosphorylation. Reduced ATP results in failure of sodium pump with influx of sodium and water leading to cell swelling. Prolonged depletion of ATP due to persistent toxic stimuli leads to disruption of protein synthetic apparatus and the cells undergo necrosis 24 . Necrosis is associated with loss of cell membrane integrity and leakage of cellular contents culminating in dissolution of cells. Nucleus become pyknotic characterized by nuclear shrinkage and increased basophilia. Atrophy in the liver cells and pyknosis of nucleus suggest parenchymal injury leading to cell necrosis 25 .

CONCLUSION
The present study revealed that haloperidol was responsible for destroying the normal architect of the developing liver. It was concluded that the liver growth was stunted as shown by poor development of hepatocytes. However, it is suggested that inspite of species differences, the antipsychotic drugs particularly haloperidol must not be given to pregnant women or must be stopped before pregnancy.