ARSENIC INDUCED HISTOMORPHOLOGICAL ALTERATIONS IN SIZE OF HEPATIC LOBULE AND AMELIORATIVE EFFECTS OF LAGENARIA SICERARIA.
Keywords: Lagenaria siceraria, Sodium arsenite, Sprague dawley.
Contact with high concentration of inert arsenic is a key health issue in various parts of the world and lays risk on millions of people in developing countries and a lot of complications in various body systems1. Water used for drinking and agricultural purposes mainly comes from ground, is the main source of arsenic contact. After ingestion, arsenic is deposited in the liver, spleen, kidneys, lungs and gastro-intestinal tract2. Arsenic is a deadly organic substance which is notorious in causing cancer, and when taken in large amounts it can be lethal3. Arsenic is added purposely in chicken feed. Wide range of chicken is fed on arsenic to enhance the bodily growth on minimum food intake and in less time period, and chicken meat gives off a vibrant and fresh look. In view of food drug administration (FDA) findings, fifty percent of screened chicken had inert arsenic in their liver.
Roxarsone, a preparation which comprises of lethal arsenic is mixed to chicken feed so that the meat gives a healthier look4. Uptake of solvable arsenic combinations is good through the gastrointestinal and respiratory tracts. Almost all of the absorbed inert arsenic undertakes methylation, chiefly in the liver, to monomethylarsonic acid and dimethylarsinic acid, which are expelled, along with remaining inert arsenic, in the urine5. Arsenic renders its toxic effects by interacting with sulfhydryl groups present in enzymes and interrupt enzymes concerning cellular respiration, which in turn is responsible for blocking glycolysis and Krebs cycle and replace phosphorus in a couple of biological processes6. Greater amount of arsenic in human body is accountable for higher levels of free radicals. These free radicals may lead to oxidative stress, hinders enzyme and mitochondrial activity7.
The liver and kidney are the prime aims for toxic effects of arsenic and the peak level of arsenic is more so in the liver compared to the kidney8.
Diversified therapeutic setups acquire the use of herbal therapies for the management and controlling of various ailments. Lagenaria siceraria has been of use in folklore prescriptions for the cure of many disorders in humans9. Excessive material, knowledge and benefits of herbal drugs is present in state of the art literature of Ayurvedic and Unani medicine. World health organization (WHO) survey reveals that majority of the residents of under developed world depend solely on old fashioned prescriptions for their vigor and vitality10. Besides these the herbs have some very essential lifesaving components used in the techniques of modern medicine. World over it is believed that traditional measures are harmless and less destructive anthropologically than man made preparations. Due to this test centers across the globe are working on plants for their organic makeup and therapeutic prospective11.
The outmoded remedial system states herbal preparations to be good for the cure of quite a range of diseases. The ayurveda has given a lot of importance to food in the treatment of diseases12.
Contemporary practitioners have even understood the worth of food stuffs, in having nutraceutical features, for the management of long standing illnesses. Lagenaria siceraria is known as Bottle gourd, an exceptional vegetable possessing all the vital elements that are obligatory for steady and virtuous well-being of human beings13. Lagenaria siceraria is conventionally utilized for its cardioprotective, cardiotonic, all purpose tonic properties and is also used as purgative and diuretic9. Protective effect onliver was studied by investigating the impact of the ethanolic extract of Lagenaria siceraria extracts against CCl4 hepatotoxicity9,14. Medicinal plants are believed to have immunomodulatory capacity of boosting the immune system of the host15. This allows the host to fight back against different attacking antigens16.
Recent, investigations have led the researchers to understand the role of this fruit in human health as it is able to provide a range of functions including anthelmintic, antibacterial, antifungal, immunomodulatory, antiallergic, analgesic, anti-inflammatory, antioxidant and hepatoprotective capabilities17.
MATERIAL AND METHODS
This randomised control trial study was approved by Ethical Committee, of the Army Medical College, Rawalpindi, to be conducted in the Department of Anatomy with National Institute of Health (NIH), Islamabad (20th March, 2017 to 14th May, 2017). Sodium arsenite was purchased from Sigma Aldrich (Product no.133027-500G). Lagenaria siceraria was purchased from local market, it was shade dried and ground to powder form then the calculated dose was given to the animals by mixing it with saline via oral gavage needle. Fifty adult male and female Sprague Dawley rats, 9-11 weeks of age and weighing 250-380gm were taken for this randomised control trial study which were divided by using probability sampling technique into five groups (control and experimental), each having 10 animals (male and females caged separately) by lottery method.
They were kept in a well-ventilated room, with temperature range of 20-26AdegC and 12h dark-light sleep cycle throughout the duration of experiment18. Rats were fed with standard laboratory diet provided by NIH. Water was provided ad libitum.Study plan and dosage is given in table-I.
Rats in groups A and B were untreated controls. Group C, D and E were given sodium arsenite 5mg/kg/day initially for a period of 4 weeks19. Animals of group A and C were sacrificed after 4 weeks and histopathological studies on liver were carried out. Within the next 4 weeks group D animals were kept without any intervention for another four weeks. Group E animals during this period of 4weeks were given sodium arsenite 5mg/kg/day and Lagenaria siceraria 100mg/kg/day (powder obtained after grinding the shade dried fruit mixed with calculated dose of normal saline)20. At the end of these 4 weeks, the animals of groups B, D and E were sacrificed, liver specimens were collected. Haematoxylin and eosin (HandE) stained sections of liver were prepared for routine histological study. Each specimen was evaluated for size of hepatic lobule. Hepatic lobular diameter was measured under low power in ten low-power fields (10X).
In each field, three lobules were selected randomly from periphery to the center of slide. Images were taken from each field with the help of Olympus digital camera (12-mega pixel). The images were then transferred to the laptop. Each image was opened in Image J v1.4821. A scale was set at 10X to measure the diameter in micrometre. Measurement tool 'Straight' was selected and the diameter to be measured was calculated by drawing a straight line. The measurements were then analysed and recorded. Results were expressed as mean for each specimen in micrometres and taken as the final reading for that specimen. Data was analysed using statistical package for social sciences version 22.
Variables were expressed as mean +- standard deviation and the significant difference was determined using one-way analysis of variance (ANOVA) followed by post Hoc Tukey's test for groups B, C, D and E. Independent sample t-test was used to find the statistical significance between the groups A and C. A p-value a(c)1/20.05 was considered significant. Mean +- standard deviation and p-value is given in table-II and III.
Table-I: Grouping of experimental study (Both male and Female Rats divided arbitrarily in following groups housed in separate cages).
Groups###Number of###Title of group###Duration of intervention###Intervention
###A###10###Control###First 4 weeks###Laboratory Diet and water ad libitum
###B###10###Control###8 weeks###Laboratory Diet and water ad libitum
###C###10###Experimental###First 4 weeks and###Sodium arsenite 5 mg /kg/day by
###sacrificed###oral gavage (Fouad et al., 2012)
###Left as such after 4 weeks of sodium
###D###10###Experimental###8 weeks and sacrificed###arsenite till the end of the experiment
###(8 weeks) without any further
###8 weeks and sacrificed###Sodium arsenite 5 mg /kg/day by
###(initial 4 weeks only###oral gavage (Fouad et al., 2012)
###E###10###Experimental###sodium arsenite and last###Lagenaria siceraria 100mg / kg body
###four weeks sodium###wt. by oral gavage (Deshpande et al.,
###arsenite and lagenaria###2008)
Table-II: Mean values of hepatic lobule diameter of control Group A and experimental group C (Independent t-test).
Parameter###Group A###Group C###p-value
Diameter of hepatic###1376.3 +- 2.05###1704 +- 2.93###<0.001*
Independent Sample T-Test
Table-III: Mean values of hepatic lobule diameter of control Group B and experimental group C, D and E (One way ANOVA followed by Post Hoc Tukey's test).
Parameter###Group B###Group D###Group E###p-value
Diameter of hepatic lobule (um)###1379.2 +- 2.82###1672 +- 1.39###1356.1 1.97###<0.001*
Post Hoc Tukey's Results
Fifty adult male and female Sprague Dawleyrats, 9-11 weeks of age, with an average weight of 284 +- 2.58gm were used in the experiment. Histologically, control groups A and B showed normal hepatic architecture with chains of hepatocytes around the central vein.The radiating cords were limited by connective tissue forming hepatic lobule 22 (fig-1a).In control group A and B, the mean diameter with SD of hepatic lobule was 1376.3 +- 2.05um and 1379 +- 2.82um. Group C showed moderate degree of disruption in hepatic architecture, characterized by the destruction of a number of liver cells and fatty change in the hepatic parenchyma leading to increase in hepatic lobular diameter23 (fig-1b). Group D had mild disruption of hepatic architecture much less as compared to group C. None of group E specimens showed disruption of the architecture. The mean diameter and SD of experimental group C increased significantly to 1704 +- 2.93um. In experimental group D, the diameter was 1672 +- 1.39um.
In e xperimental group E, the mean diameter and SD was measured to be 1356.1 +- 1.97um. While comparing the size of hepatic lobule the maximum increase was seen in group C which reduced in group D and reverted back to nearly normal size in group E specimens. Bar charts were also plotted for the values of mean hepatic lobular diameter between group A and C, groups B, C, D and E (fig-2 and 3). On intergroup comparison of control groups, A and B with the experimental groups C, D and E, the results were statistically highly significant (p-value<0.001*).
Exposure to sodium arsenite through various routes has adverse effects on various tissues and organs, leading to morbidity24. Accordingly, this study was designed to investigate the effect of sodium arsenite on the liver of adult rats and whether subsequent administration of lagenaria siceraria can alter the effects. In present study, acute exposure to mild doses of sodium arsenite altered the liver architecture with fatty change in the lobules affecting their size. In control group A and B the architecture was normal with no fatty change and disruption of architecture and the size of lobule was in the normal limits. Group C showed severe degrees of disruption of architecture culminating in increase in dimensions of hepatic lobule25. Group D showed mild increase in size of hepatic lobule. Group E showed a picture like that of the control group A and B. Histological changes were highly significant when inter group comparison was done between A, B, C, D and E.
These results were also studied on bar graphs showing comparison of size of hepatic lobule in group A and C (fig-2), and between B, C, D and E (fig-3). These results were in accordance with the previous works26,27. Oxidative stress, apoptosis, and upregulation of transcription factors such as AP-1, ATF-2, and Elk-1 are the prospective target sites for arseniteinduced nephrotoxicity and hepatotoxicity. Lagenaria siceraria when chemically studied, it was found to be composed of flavonoids. Flavonoid containing plants have influence on arachidonic acid metabolism and are supposed to have anti-inflammatory, cardio protective and anti-allergic effect. Heavy metals can lead to oxidative stress and lead to hepatotoxicity. Lagenaria siceraria has marked oxidative potential and able to revert this oxidative stress and is able to reduce the fatty infiltration in the interstitium and periportal areas.
In the current study also when lagenaria siceraria was given to the animals after exposure to heavy metal toxicity the disruption of lobules of liver and the increase in their size was reduced (table-II and III). Bar charts were also plotted to study the intergroup comparison. Mechanism behind the hepatic toxicity is that most absorbable component seventually go to the liver for breakdown. This puts up the liver to severe deadliness28. Enzymes like aspartate aminotransferase and alanine aminotransferase are found chiefly in liver tissue and cellular injury as a result of arsenic exposure releases these enzymes into the blood stream and the plasma levels of these enzymes specify hepatotoxicity29. Epidemiological studies have obviously showed an association between chronic arsenic exposure and abnormal liver function, hepatomegaly, hepatoportal sclerosis, ascites, liver fibrosis and cirrhosis from exposure to arsenic in the drinking water.
Our study suggests that sodium arsenite caused disruption of hepatic architecture with fatty change leading to increase in size of hepatic lobule in adult rats while collateral intervention with Lagenaria siceraria had protective effect on the hepatic architecture and subsequently on the size of hepatic lobule.
We are grateful to our colleagues for their persistent sustenance and all the people in Anatomy Department, Army Medical College, Rawalpindi for their valuable input.
Saima Qureshi conceived the idea, created the manuscript, Umbreen Noor and Ayesha Baqar analysed the data. Dr Khadija Qamar did the critical analysis and revised the manuscript.
CONFLICT OF INTEREST
The authors of this study reported no conflict of interest among their idea.
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|Publication:||Pakistan Armed Forces Medical Journal|
|Date:||Feb 28, 2019|
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